A subtype of human papillomavirus 5 (HPV-5b) is closely associated with carcinomas in the disease epidermodysplasia verruciformis (EV). The complete genome was cloned from virus particles in benign lesions of a patient with EV and sequenced: it was 7779 nucleotides long and consisted of six open reading frames (ORFs) (E6, E7, E1, E2, E4, and E5) in the early region, three ORFs (L2, L3, and L1) in the late region, and a noncoding region, all existing on one DNA strand. The 40% segment of the HPV-5b genome specifically amplified in carcinomas was cloned from a primary carcinoma of the same EV patient and sequenced: it was 3143 nucleotides long and corresponded to a segment of the original HPV-5b genome containing the entire sequences of E6, E7, and the noncoding region and portions of E1 and L1. Compared to the whole genomic DNA, no mutations were detected in this probable malignancy-associated viral subgenomic segment cloned from carcinoma. These results suggest that amplification of the viral segment containing E6, E7, and the noncoding region may play a role in the malignant conversion of HPV-5b-infected benign lesions and that mutations in these genes or regions are not necessarily required.