A polymerase chain reaction (PCR) was used to detect herpes simplex virus (HSV) deoxyribonucleic acid (DNA) in CSF of 109 patients with possible herpes simplex encephalitis. HSV DNA was found in 20/109 patients. In 14 of these patients the diagnosis was confirmed by a rise in CSF antibodies, isolation of HSV from the brain, or both. In 3 patients CSF antibodies did not rise and 3 patients did not have a follow up lumbar puncture or a brain biopsy. In 19/20 patients HSV DNA was present in the first CSF specimen. The virus was identified as HSV I in 15 patients and HSV II in 4; the virus was not typed in the other patient. A possible diagnosis of herpes simplex encephalitis was not confirmed in the 89 PCR-negative patients. HSV DNA was present in CSF of 3 patients who had meningitis with herpetic genital infections but it was not found in 24 patients with other neurological diseases. The results suggest that the detection of HSV DNA in CSF using a PCR assay will be an accurate method of early diagnosis of herpes simplex encephalitis.