Cerebrospinal fluid (CSF) and serum specimens from patients with presumed herpes simplex encephalitis (HSE) were characterized for antibodies to herpes simplex virus (HSV) by immunoblot and other immunoassays. Specimens from patients proven to have HSE (biopsy-proven) were compared with those from patients with other diseases diagnosed by brain biopsy (biopsy-negative for HSV). Immunoblot of CSF demonstrated that antibodies to HSV-specific polypeptides, particularly to glycoprotein B, were present in a matched serum specimen. When purified glycoprotein B was used to detect CSF antibodies, 34 of 35 specimens from biopsy-proven patients were reactive (97% sensitivity) compared with only six of 22 specimens obtained from biopsy-negative patients (73% specificity). If leakage of antibodies to a marker virus (adenovirus) was determined and controlled, the specificity increased to 100%. These diagnostic assays provide useful tools for the retrospective assessment of CSF specimens obtained from patients with presumed HSE. © 1987 by The University of Chicago.