A nested polymerase chain reaction (PCR) was evaluated for the detection of cytomegalovirus (CMV) DNA in cerebrospinal fluid (CSF). CSF and serum samples from 19 AIDS patients with intracerebral CMV infection diagnosed at autopsy were retrospectively examined. As controls, CSF and serum samples from 15 AIDS patients with only extracerebral CMV involvement at autopsy, from 10 AIDS patients without CMV infection at autopsy, and from 10 anti-human immunodeficiency virus-negative patients without ongoing CMV infection, were studied. CMV DNA was detected from patients with intracerebral CMV infection in 9 of 9, 5 of 6, and 1 of 4 CSF samples collected, respectively, 1-30, 30-90, and 90-300 days before death. Twelve of 13 sera from these patients were CMV PCR-positive. None of the control patients had CMV DNA in CSF. PCR was positive in 6 of 8 sera from AIDS patients with only extracerebral CMV infection and in serum from 1 AIDS patient without CMV involvement at autopsy. CMV PCR on CSF is highly sensitive and specific. It should be considered a rapid and reliable diagnostic method for CMV infection of the central nervous system.