The fleet left the port of Shimonoseki in southern Japan on Sunday.
The whalers plan to kill up to 50 humpback whales in what is believed to be the first large-scale hunt for the once-nearly-extinct species since a 1963 moratorium in the Southern Pacific put them under international protection.
The mission also aims to kill as many as 935 minke whales and up to 50 fin whales in what Japan's Fisheries Agency says is its largest-ever scientific whale hunt. The expedition lasts through April.
The environmentalist group Greenpeace said its ship, the Esperanza, was searching for the fleet south of Japanese territorial waters and would shadow the ships to the Southern Pacific.
"It's a large ocean, but we're going to track them down," expedition member Dave Walsh told The Associated Press by telephone.
Greenpeace claimed the fleet had turned off radio transponders that signal its location. Hideki Moronuki, head of the whaling section at Japan's Fisheries Agency, denied that.
Anti-whaling nations have widely criticized the hunt.
New Zealand's Prime Minister Helen Clark said the fleet should have never sailed.
"It would just be better if the Japanese stayed home and didn't come down under the guise, the deception, the claim that it is scientific whaling when they want to take a thousand whales," she told New Zealand's TV One.
The Australian government was "deeply disappointed" by the fleet's departure, Foreign Minister Alexander Downer and Environment Minister Malcolm Turnbull said in a statement. "The government again appeals to Japan to reconsider its position on this inhumane practice."
At a farewell ceremony for the expedition, the head of the mission said Japan should not give in to the demands of whaling opponents and criticized environmental groups' tactics.
"They're violent environmental terrorists," Hajime Ishikawa said to a crowd on the dock. "Their violence is unforgivable. ... We must fight against their hypocrisy and lies."
The conservation group Sea Shepherd harried Japan's fleet in the last Antarctic hunt in February. Japan's government released video of protesters launching smoke canisters from a Sea Shepherd ship and dropping ropes and nets to entangle the Japanese vessels' propellers.
The hunt ended early after a fire broke out aboard the mother ship, killing one crew member and forcing the fleet back to port. It was not clear what caused the blaze.
Fisheries Agency officials refused to say whether any special safety precautions were being taken for the current hunt.
Japan says that it needs to kill whales to conduct research on their reproductive and feeding patterns, and that the number it kills is far too small to endanger their overall populations. The International Whaling Commission allows scientific whale hunts, but critics say Japan uses science as a cover for commercial whaling.
An IWC moratorium on commercial whaling took effect in 1986. Japan has killed almost 10,500 mostly minke and Brydes whales under research permits since then.
The head of Japan's Fisheries Agency, Shuji Yamada, said Tokyo's scientific research would help prove that sustainable whaling is possible.
The American Cetacean Society estimates the humpback population has recovered to about 30,000 to 40,000, but the World Conservation Union lists the species as "vulnerable."