In a retrospective review of data from 168 patients with suspected progressive multifocal leukoencephalopathy (PML) between 1996 and 2006, JC virus (JCV) PCR on cerebrospinal fluid (CSF) samples was positive only in human immunodeficiency virus (HIV)-infected patients with low CD4 cell counts and in severely immunocompromised patients with radiographic lesions consistent with PML or infectious processes generally. Of note, one HIV patient with a very low CD4 cell count had a positive JCV PCR despite a normal magnetic resonance imaging exam. We concluded that JCV PCR testing on CSF specimens should therefore be targeted to these high-risk patients.