The ordinance requires them to pass a history test and get a license before speaking to groups about the history of the Liberty Bell, Independence Hall and other landmarks.
Mayor Michael Nutter signed the law in April amid concerns some guides were perpetuating gross inaccuracies in Philadelphia's history.
The guides, backed by a public-interest law firm, argue the city has gone too far and want the law overruled.
They say their constitutional rights are being violated in the very city where the Declaration of Independence was adopted 232 years ago.
A federal judge will hear the lawsuit.
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