Specific human papillomavirus (HPV) types are strongly associated with intraepithelial neoplasia and invasive cancer of the cervix. In contrast, the role of HPVs in the pathogenesis of invasive carcinoma of the vulva is poorly understood. We have employed in situ hybridization for the detection of subgenomic transcripts in four vulvar specimens to elucidate the role of HPV type 16 in the development and progression of vulvar cancer. These analyses revealed that the transcripts of the E6-E7 region were more abundant than those of the L1-L2 region in vulvar neoplastic tissues. The transcripts from early and late region of HPV-16 continued to increase with the differentiation of the epithelial cells in both the warty and the basaloid types of vulvar precancerous lesions. This pattern persisted in invasive warty carcinoma but not in basaloid invasive carcinoma; the transcripts in basaloid carcinoma were distributed in an even and discrete pattern. In contrast to earlier studies, L1-L2-region transcripts, as well as viral capsid protein, were detected in focal areas of well-differentiated cells of invasive warty carcinoma. These findings suggest that expression of HPV-16 is regulated by the degree of cellular differentiation.