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A herpes simplex virus type 2 (HSV 2) etiology was sought in 93 consecutive cases of herpes simplex encephalitis (HSE) in immunocompetent post neonate patients. Antibodies to HSV 2 glycoprotein G antigen were determined by an enzyme-linked immunosorbent assay (ELISA) and HSV 2 DNA in cerebrospinal fluid (CSF) by a nested polymerase chain reaction (PCR) assay with primer pairs in the glycoprotein G gene. Evidence of HSV 2 infection was found in 6 patients; HSV 2 DNA was demonstrated in CSF and the intrathecal HSV 2 antibody response confirmed the findings. Five of the 6 patients with HSV 2 encephalitis presented a clinical picture, CSF, EEG, and CT findings characteristic of severe HSE. An atypically mild clinical course was seen in one patient. HSV 2 should be considered as an etiological agent in the viral diagnosis of HSE. With a combination of nested PCR assays for HSV 1 (primer pairs in the glycoprotein D gene) and HSV 2 in 10 microliters of CSF with no other preparation than freeze-thawing, HSV 1 or HSV 2 DNA was detected in 88 out of 93 (95%) of the first CSF specimens collected after the onset of neurological HSV disease. These findings extend and confirm previous results with PCR as a rapid and sensitive tool for early diagnosis of HSE.