We studied the presence of human papillomavirus (HPV) DNA by in situ hybridization in 74 colposcopically defined subclinical vulvar lesions obtained from 51 patients (mean age 25 years, range 18-42 years) referred for atypical Papanicolaou smears to the Colposcopy Clinic. Serial sections of formalin-fixed, paraffin-embedded biopsy tissue were stained by routine hematoxylin and eosin stain or were separately hybridized with biotin-labeled probes of HPV types 6, 11, 16, and 18 at high stringency and with mixed HPV probes at low stringency. Histologic evidence of HPV infection (koilocytosis or dysplasia) was found in eight lesions. Overall, 10 lesions were positive by in situ hybridization with HPV DNA. Although certain colposcopic features were more strongly associated with HPV DNA than others were, we were unable to demonstrate any consistent correlation between HPV DNA positivity or specific HPV DNA types and colposcopic categories. There was little correlation between HPV DNA positivity and the histopathologic incidence of HPV infection and dysplasia. Three of the 10 hybridization-positive lesions were severely dysplastic as compared with 0 of 64 nonhybridizing lesions. In spite of this association between HPV DNA and dysplasia, HPV DNA hybridization added no additional clinically relevant information to the histopathologic findings. We emphasize the significance of careful colposcopic examination of the vulva with directed biopsies.