Cerebrospinal fluid (CSF) specimens were obtained from patients with presumed herpes simplex encephalitis who underwent brain biopsy for diagnostic confirmation. Coded CSF specimens were fixed on nitrocellulose filter paper and probed with a pool of monoclonal antibodies directed against four herpes simplex virus glycoproteins (gB, gC, gD, and gE). Herpes simplex virus antigen was detected in 35 of 40 specimens obtained from 26 biopsy-positive patients. In contrast, only three of 25 specimens from 17 biopsynegative patients gave positive results by this assay. An additional 30 CSF specimens from patients with proven bacterial and fungal infections were all negative by this assay. For all specimens tested, the sensitivity and specificity of the assay were both 88070. However, when results were evaluated by patient, the sensitivity was 92% (24 of 26) with a specificity of 82% (14 of 17). Among specimens collected one week or later after disease onset, the sensitivity was 100%, with a specificity of 93%. © 1987 by The University of Chicago.