Karadzic faces a total of 11 charges at the U.N.'s Yugoslav war crimes tribunal. Judge Iain Bonomy, who presided over the hearing, entered not guilty pleas on Karadzic's behalf.
He is charged with genocide for allegedly masterminding atrocities, including the slaughter of more than 8,000 Bosnian Muslims in Srebrenica in July 1995 and the deadly siege of Sarajevo, when he was president of the breakaway Bosnian Serb republic.
"This court is representing itself falsely as a court of the international community whereas it is in fact a court of NATO whose aim is to liquidate me," Karadzic said. "I will not plead, in line with my standpoint as regards to this court."
The next hearing is Sept. 17 when Karadzic said he would challenge the court's jurisdiction. No trial date has been set.
Karadzic, 63, was back in court for only his second public appearance since his arrest July 21 in the Serb capital Belgrade after 13 years on the run.
The 25-minute hearing was a crucial step along the path to Karadzic's trial for allegedly masterminding the worst atrocities perpetrated by Serb forces in the 1992-95 Bosnian war, which claimed the lives of an estimated 100,000 people.
Karadzic, looking less tired than at his first appearance shortly after his arrest a month ago, employed sarcasm that failed to amuse the Scottish judge.
When Bonomy told the former leader he was entering not guilty pleas, Karadzic said: "May I hold you to your word ... that I am not guilty?"
Bonomy replied: "We shall see in due course, Mr. Karadzic."
Outside the court, the president of the Mothers of Srebrenica group that represents survivors of the massacre said she wanted to witness the case.
"We came to see (the) initial appearance of the biggest butcher of the 20th century in the Balkans," Munira Subasic said.
Prosecutors accuse Karadzic of orchestrating a savage campaign of ethnic cleansing to drive Muslims and Croats out of territory claimed by a breakaway Bosnian Serb ministate.
According to his indictment, the reign of terror began with the destruction of villages and establishment of brutal internment camps where civilian detainees were tortured, raped and murdered.
It progressed through the horror of the 44-month siege of Sarajevo, during which Serb forces relentlessly shelled the Bosnian capital and sniped at its inhabitants as they sat in trams, stood in line for bread and even as they mourned at funerals.
Srebrenica was its murderous climate - Europe's biggest massacre since the Holocaust.
Bonomy pressed prosecutors to work fast to draw up a new, streamlined indictment against Karadzic.
Prosecutor Alan Tieger said he hoped to have a new charge sheet ready by the end of September.
"I sincerely hope you are not serious about that date," Bonomy told Tieger.