Laboratory procedures were compared with brain biopsy findings in 113 biopsy-proven patients with herpes simplex virus (HSV) encephalitis and 93 biopsy-negative individuals. Examinations of brain tissue by histopathology, immunofluorescence, and electron microscopy demonstrated evidence of HSV infection in 56%, 70%, and 45% of proven cases and apparently false-positive results in 14%, 9% and 2% of those biopsy-negative. Serologic assessments revealed that HSV encephalitis occurred as both a primary (30%) and recurrent (70%) infection. Among patients with HSV encephalitis, 28% failed to seroconvert or seroboost within one month of the onset of disease. Titers of passive hemagglutinating and IgG immunofluorescent antibodies increased fourfold in the cerebrospinal fluid in 74% and 94%, respectively, of patients with proven disease. Similar percentages of patients had antibody ratios in serum and cerebrospinal fluid of less than 20 over the same interval. These data indicate the need for development of noninvasive diagnostic procedures.