To determine the diagnostic value of the polymerase chain reaction (PCR) in establishing the rapid diagnosis of herpes simplex virus encephalitis (HSE) in the pediatric age group, we performed PCR to detect herpes simplex virus (HSV) in the cerebrospinal fluid of 8 neonates with HSV infection (4 with central nervous system involvement), 11 infants and children with suspected HSE (4 proved, 1 presumed, 6 excluded), and 105 control patients who had cerebrospinal fluid obtained as part of the evaluation for other diagnoses. The HSV DNA was amplified and typed by using primers specific for the DNA polymerase gene of HSV types 1 and 2. Herpes simplex virus DNA was detected in the cerebrospinal fluid of 3 of 4 neonates with CNS involvement (all with HSV type 2) and 3 of the 4 patients with proved HSE (all with HSV type 1). No HSV DNA was detected in the 4 neonates without CNS disease, the 1 patient with presumed HSE, the 6 patients who had HSE excluded from the diagnosis, and the 105 control patients. Overall, HSV PCR had a sensitivity of 75%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 98%. These results indicate that PCR is a useful noninvasive test in establishing the diagnosis of acute HSE, but a negative result did not exclude the diagnosis.