Speaking by video link from Belmarsh Prison in southeast London, Assange said: "I do not wish to surrender myself for extradition for doing journalism that has won many awards and protected many people."
His formal refusal to be extradited marks the start of what is expected to be a bruising legal battle over whether he will be brought to trial in the United States.
Assange, wearing jeans and a sports jacket, appeared calm during the brief hearing at London's Westminster Magistrates' Court. Some of his supporters who couldn't get seats in the small courtroom chanted support for Assange from the hallways, shouting "Shame on you" at the judge.
Judge Michael Snow said it would likely be "many months" before a full hearing was held on the substance of the U.S. extradition case. The judge set a procedural hearing for May 30, with a substantive hearing to follow on June 12 once a full U.S. extradition request has been received and studied by Assange's lawyers.
Legal experts predict it will likely take 18 months or longer to resolve the case, with each side able to make several appeals of unfavorable rulings.
That extradition request is no longer active, but Swedish officials say the rape investigation may be revived now that Assange is no longer out of reach in the Ecuadorian Embassy.
Assange says he sought asylum in the embassy because he feared being sent to the U.S. to face charges related to WikiLeaks' publication of classified U.S. military documents.
Manning served several years in prison for leaking classified documents to WikiLeaks. She was jailed again in March after refusing to testify to a grand jury investigating the secret-spilling organization.
Ben Brandon, a lawyer representing the U.S. government, said in court Thursday that U.S. investigators had obtained details of chatroom communications between Manning and Assange in 2010. Brandon said the pair had "engaged in real-time discussions regarding Chelsea Manning's dissemination of confidential records to Mr. Assange."
He said the documents allegedly downloaded from a classified U.S. computer included 90,000 activity reports from the war in Afghanistan, 400,000 Iraq war-related reports, 800 Guantanamo Bay detainee assessments and 250,000 State Department cables.
The U.S. charge against Assange carries a maximum five-year prison sentence, but Assange is worried the U.S. could add further, more serious allegations against him.
"The fight has just begun. It will be a long one and a hard one," said WikiLeaks editor-in-chief Kristinn Hrafnsson, who claimed Assange was being held in "appalling" conditions at Belmarsh Prison.
He said Assange was confined to his cell 23 hours a day, "what we call in general terms solitary confinement."
A few dozen WikiLeaks supporters holding signs reading "Free Assange" and "No extradition" gathered outside the London courthouse before Thursday's hearing.
Some who had waited for two hours hoping to get in were bitterly disappointed when those seats were filled by journalists and lawyers. They shouted angrily at court staff and complained they were being discriminated against for backing Assange. Some later blocked a busy main road outside the court, bringing traffic to a halt.
Assange was arrested last month in London after his relationship with his embassy hosts went sour and Ecuador revoked his political asylum.
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