We examined lesions from six women with multifocal neoplasia of the lower genital tract (vulva and cervix or vulva and vagina) for the presence of human papillomavirus (HPV) DNA sequences by DNA-DNA in situ hybridization. Cervical tissue specimens from four of the five women with carcinoma in situ (CIS) of the cervix were found to contain HPV type 16 DNA sequences. Vulvar lesions from three of these women also contained HPV-16 DNA and were histologically consistent with CIS. In each instance, hybridization with HPV-16 DNA probes was seen only in areas of the epithelium that contained evidence of surface maturation or koilocytotic atypia (KA). The HPV DNA was not visualized in regions of these lesions in which the full thickness of the epithelium was occupied by poorly differentiated cells. Virus capsid antigens were only detected in a very few cells in two of the four cervical lesions that contained HPV-16 DNA. HPV type 16, which has consistently been associated with the development of cervical cancer, is further implicated as an agent in the pathogenesis of genital cancers by demonstration of the virus genome in neoplasia at nonadjacent sites.