A single pair of oligonucleotide primers selected within a highly conserved region of the DNA polymerase gene of the herpesviruses was designed to amplify related viral genomes, i.e., herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, and cytomegalovirus, by the polymerase chain reaction. A simple restriction enzyme analysis of these amplified products allowed accurate characterization of the herpesvirus type. Ninety-nine cerebrospinal fluid samples from 36 patients (including newborns, children, and adults) with acute encephalitis were tested for the presence and identification of herpesvirus DNA by this approach. High levels of viral DNA, which were readily visualized by simple ethidium bromide staining, were found in all these patients from the first days of the disease and, in some cases, until the third week following the onset of acute encephalitis. The herpesvirus type was rapidly identified by enzymatic digestion in 33 patients' samples and by hybridization and direct sequencing in the last 3 patients' samples. Our results show that the polymerase chain reaction provides a highly sensitive and specific technique for the identification of herpesviruses DNA in cerebrospinal fluid that should be of value for early and rapid diagnosis, therapeutic decisions, prognostic evaluation, and epidemiological studies.