AbstractHerpes simplex virus (HSV) infection constitutes an immediate threat to the neonate of pregnant women who deliver vaginally, and thus requires a rapid, specific means of diagnosis that is easily applicable to cervicovaginal smears. We applied the peroxidase‐antiperoxidase technique to variously fixed, previously stained Papanicolaou smears from 60 women with HSV and 18 negative controls using an HSV‐2 antibody and either diaminobenzidine (DAB) or aminoethylcarbazol (AEC) as the chromogen and Mayer's, Gill's, or Lillie‐Mayer's hematoxylin as the counterstain. With Papanicolaou stain alone, there was adequate cytologic demonstration of either single cells in aggregates (11%), syncytial giant cells (40%), or both (49%) that displayed a ground‐glass appearance (68%), discrete nuclear inclusions (5%), or both (28%). With the peroxidase‐antiperoxidase technique, 57 of the 60 HSV specimens (95%) stained moderately or strongly positive for HSV‐2, as did sections of herpetic encephalitis and esophagitis. There was no false‐positive staining in any of the 18 control smears revealing koilocytosis, Chlamydia, or nonspecific vaginitis. Positivity of the immunostain was more vivid and evenly dispersed throughout the cytoplasm with AEC than with DAB, but tended to wash away with alcohol‐based counterstaining. In contrast, DAB was more stable, but was positive predominantly at the cell periphery and cytoplasmic processes. Lillie‐Mayer's stain provided the best counterstaining for the cytologic visualization of virocytes and accompanying inflammatory and epithelial cells, which revealed a minimal degree of atypia. The fixative used had no influence on the frequency or degree of immunopositivity of virocytes, but wet fixation with 95% alcohol or Carbowax led to less background staining than Spray‐Cyte®. The technique is sensitive, specific, reproducible, more cost‐effective, and less time‐consuming than the alternate methods of immunofluorescence, viral culture,