Cell monolayers were inoculated with 169 fresh and 76 previously frozen clinical specimens and examined for the presence of herpes simplex virus (HSV) by noting the appearance of characteristic cytopathic effect (CPE) and by using a direct immunoperoxidase (IP) stain for viral antigen. HSV was detected by IP staining in 40 of 169 (23.7%) monolayers and by CPE in 39 of 169 (23.1%) monolayers inoculated with fresh specimens. All 40 isolates were detected and confirmed by IP staining within 24 h. Although 39 of 40 isolates were detected by CPE, only 9 of 39 (23%) were positive within 24 h. CPE was observed at 2.7 days on the average, but 4 days were required before 90% of the cultures were positive and more than 5 days were required before all HSV isolates were recognized. Similar results were observed for frozen specimens. HSV was detected earlier with IP staining, which demonstrated more extensive infection of cell monolayers inoculated with titrated fresh culture isolates and clinical specimens than did CPE. IP staining reduces the amount of time required for detection and identification of HSV in culture, is readily adaptable for use in the clinical laboratory, and permanent stained preparations can be made.