Detection of human cytomegalovirus (HCMV) pp65 in cerebrospinal fluid (CSF) polymorphonuclear leukocytes (PMNL) was investigated in 32 CSF samples from 25 AIDS patients with neurologic symptoms. HCMV pp65-positive PMNL were detected in 9 CSF samples from 3 patients with polyradiculoneuropathy (frequency, 1.2%-20.8%) and 1 with encephalitis (frequency, 56%). pp65-positive PMNL appeared hypersegmented compared with peripheral blood PMNL. HCMV was isolated from 3 CSF samples from 2 patients with polyradiculoneuropathy. Retrospective quantitation of viral DNA by polymerase chain reaction (PCR) was in agreement with pp65 detection in 27 of 28 samples tested (96.4% concordance). Specificity and sensitivity of pp65 assay versus PCR were 100% (17/17 negative samples) and 90.9% (10/11 positive samples), respectively. Examination of CSF for presence of pp65-positive PMNL appears to be a sensitive and specific test for diagnosis of HCMV infections of the nervous system.