The vote was 47-51.
Moments earlier, the Senate had agreed to make one relatively minor change in the law, voting to strip out a paperwork requirement for businesses.
President Barack Obama, who has vowed to veto any total repeal of his signature legislative accomplishment, has said he would accept the change. It does not directly affect health care.
Republicans conceded in advance their attempt at total repeal would fall short. But they also said they had accomplished an objective of forcing rank and file Democrats to take a position on an issue that reverberated in the 2010 campaign and may play a role in 2012.
Senate Republican leader Mitch McConnell said the vote marked an opportunity for Democrats who voted for the bill last year "to listen to those who have desperately been trying to get your attention."
"To say, yes, maybe my vote for this bill was a mistake, and that we can do better," McConnell said.
Democrats worked to minimize any political repercussions, a concern for a party already acutely aware it must defend 23 seats - and its shrunken Senate majority - in the 2012 elections.
Majority Leader Harry Reid, D-Nev., said the Republican repeal movement would "take away a child's right to get health insurance and instead give insurance companies the right to use asthma or diabetes as an excuse to take away that care."
"It would kick kids off their parents' health insurance," Reid said. "It would take away seniors' rights to a free wellness check."
Democrats also countered with the proposed repeal of the law's requirement that businesses, charities, and state and local governments file income tax forms every time they purchase $600 or more in goods.
It was approved 81-17, after Republicans pointed out it had originally been their idea.
Across the street from the Capitol, Democrats convened a Judiciary Committee hearing to solicit testimony on the constitutionality of the law they passed and Obama signed months ago.
"Many who argue the Affordable Care Act is unconstitutional are the same people who condemn judicial activism," said Sen. Dick Durbin, D-Ill., who presided. "They are pushing the Supreme Court to strike down this law because they could not defeat it in Congress."
Republicans were scathing in response.
"The sensible process would have been to have . held a hearing on the law's constitutionality before the bill passed, not after," said Sen. Chuck Grassley of Iowa. "Like Alice in Wonderland, sentence first, verdict afterward."
Two federal judges have ruled the law is unconstitutional, partially or in its entirety, citing a requirement for individuals to purchase coverage and pay a penalty in taxes if they fail to do so. Two other judges have upheld the law.
The controversy has yet to reach the Supreme Court, but it is widely expected to, and Sen. Bill Nelson, D-Fla., announced he would file legislation urging the justices to act quickly.
The maneuvering occurred around a law as ambitious as any in recent years, and as controversial. According to the Congressional Budget Office, it would expand coverage to tens of millions who lack it, crack down on insurance industry abuses and cut federal budget deficits. At its core, the bill would require most Americans to purchase insurance, a so-called individual mandate that has become one of the principal points of opposition among Republicans and the tea party activists who propelled them to gains last fall.
The bill's critics argue the law gave government too large a role in the health care system, will harm Medicare and raises taxes and fees that will burden the economy. They also sharply dispute the CBO estimate that deficits will fall once the bill takes effect, arguing that the forecasts rest on spending cuts to Medicare and other programs that will not materialize.
Either way, the day's events shaped up as the latest maneuvering in a struggle that has spanned more than two years.
Republicans said a proposal by Sen. Debbie Stabenow, D-Mich., to eliminate the reporting requirement to the Internal Revenue Service was legislative pilferage, noting that Sen. Mike Johanns, R-Neb., filed a bill to that effect last year.
The measure calls for $44 billion in spending cuts to offset the revenue loss from the change, but unlike Johanns' earlier measure, Stabenow's specifies that none of the funds can come from Social Security.
Under federal law, Social Security benefits are generally guaranteed. As a result, the provision Stabenow advanced assures merely that no administrative costs can be cut at the agency.
No similar protection was included for the agency that oversees Medicare.
The House approved legislation repealing the health care law last month on a party-line vote, ignoring a veto threat from Obama and Reid's blunt statement the bill would never see the light of day in the Senate. McConnell responded quickly that he would look for an opportunity to force a vote.
All 47 Republicans voted to repeal the law, but no Democrat joined them.
At a news conference shortly after the vote, Sen. John Cornyn of Texas, the chairman of the party's campaign committee, said the vote marked the "first steps in a long road that will culminate in 2012 whereby we will expose the flaws and the weaknesses in this legislation."
The law that passed a year ago had the support of 58 Democrats and two independents aligned with them. All 40 Republicans voted against it.
Democratic ranks have been thinned since then, and their current majority is 53-47.
Of those 53 seats, 23 are on the ballot in 2012, including several that Republicans are targeting. One on the list, Sen. Ben Nelson of Nebraska, said in advance he would oppose the Republican repeal proposal.
"There are a lot of good parts in the bill and some that I will work to improve," Nelson told reporters in his home state. "The repealers already have health care. But they're ready, willing and eager to take it away from hundreds of thousands of Nebraskans."
Associated Press writer Laurie Kellman contributed to this report.