Vulvar biopsies and explants from patients with vulvitis, hyperkeratosis, condyloma acuminatum, severe dysplasia, and squamous cell carcinoma were screened for herpes simplex virus type 2 (HSV-2) antigens. Immunoperoxidase staining for VP143, an early nonstructural polypeptide of HSV-2, was identified within three biopsies exhibiting severe dysplasia, three of seven with carcinoma in situ, and one of three with invasive squamous cell carcinoma. Similar staining for VP143 was observed in derived explants. Staining for VP119, the major envelope glycoproteins of HSV-2, was identified within tissues which were positive for VP143. Both proteins were expressed in the absence of staining for virus capsid proteins, detection of virus structures by electron microscopy, and isolation of infectious virus by co-cultivation, indicating that only a fragment of the virus genome was expressed. Neither VP143 nor VP119 was identified in biopsies exhibiting vulvitis, hyperkeratosis, or condyloma acuminatum. These data indicate a close relationship between HSV-2 and vulvar neoplasia.