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The treason charges relate to Hichilema's convoy allegedly refusing to give way to President Edgar Lungu's motorcade on a main road in the west of the country at the weekend.
Hichilema's arrest has fanned political tensions in Zambia, where the United Party for National Development (UPND) refuses to accept last year's election result when President Lungu retained power.
Hichilema, known as "HH", has been denied access to lawyers, his party said, calling for supporters to protest outside court at the hearing due on Tuesday.
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"We are demanding the immediate and unconditional release of HH as he has not committed any offence," UPND deputy Geoffrey Mwamba told a news briefing in Lusaka.
Police accuse Hichilema of putting the president's life in danger and "unreasonable, reckless and criminal" conduct in not pulling off a narrow road to make way for him.
Video footage showed vehicles travelling at high speed and nearly colliding as the presidential motorcade edged past Hichilema's convoy.
"Hakainde Hichilema has now been denied access to see his lawyers, which is a basic human right," party spokesman Charles Kakoma said in a statement.
Treason is a non-bailable offence in Zambia, with a minimum jail term of 15 years and a maximum sentence of the death penalty.
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Hichilema and five party aides have also been charged with breaking the highway code and use of insulting language.
The UPND criticised the raid as "barbaric" after Hichilema's wife and children were trapped inside and choked by tear gas.
Hichilema has launched several legal attempts to challenge the result of the August election, which he lost narrowly.
He says that the vote was rigged and accuses Lungu of political repression in Zambia, which has been known for its relative stability.
The election campaign was marked by clashes between supporters of Lungu's Patriotic Front (PF) party and the UPND.
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Hichilema, a wealthy self-made businessman, has run unsuccessfully for president five times.
The UPND called on supporters not to protest on Thursday as it said the government was seeking a chance to impose a state of emergency.
Some supporters at the press conference carried placards reading "Release HH before Zambia goes into flames".
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