In order to establish the role of human papillomavirus (HPV) in carcinogenesis of epidermodysplasia verruciformis (EV), the presence, the molecular characteristics and the physical state of HPV DNA in a benign lesion, a primary carcinoma and a metastatic carcinoma developing in the same EV patient were studied and compared. Of the 2 HPV DNAs isolated from benign macular lesions, only one (a subtype of HPV 5) was detected in both primary and metastatic tumors. Only one normal species of viral DNA molecule was detected in the benign lesion, whereas most, if not all, viral DNA molecules present in the carcinoma (both primary and metastatic) were aberrant ones. The major viral DNA molecule in the primary carcinoma was a large HPV DNA with duplicated 40% subgenomic segments, and was present as free episomes. The major viral DNA molecule in the metastatic carcinoma was the 40% subgenomic segment itself, lacking the remaining 60% segment of the viral genome, and was integrated within cellular DNA. Thus, HPV DNA was present in tumors at any stage of malignancy, and its molecular characteristics and physical state changed not only with the development but also with the enhancement of malignancy, consistently conserving its defined 40% subgenomic segment as the predominant viral sequences. Our results suggest that HPV 5 may be actually involved in carcinogenesis in EV patients.