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The human polyomavirus, JCV, causes the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised patients. We found that the serotonergic receptor 5HT2AR could act as the cellular receptor for JCV on human glial cells. The 5HT2A receptor antagonists inhibited JCV infection, and monoclonal antibodies directed at 5HT2A receptors blocked infection of glial cells by JCV, but not by SV40. Transfection of 5HT2A receptor–negative HeLa cells with a 5HT2A receptor rescued virus infection, and this infection was blocked by antibody to the 5HT2A receptor. A tagged 5HT2A receptor colocalized with labeled JCV in an endosomal compartment following internalization. Serotonin receptor antagonists may thus be useful in the treatment of progressive multifocal leukoencephalopathy.