Human papillomavirus type 6 (HPV 6) DNA was detected in an episomal form in DNA extracted from an invasive squamous carcinoma of the vulva. The viral DNA, designated HPV6-T70, was molecularly cloned. Restriction analysis of the HPV6-T70 genome revealed an insertion of approximately 35 bp in the 5' portion of the upstream regulatory region relative to the prototype HPV 6b genome, cloned from a benign genital wart (E.-M. de Villiers, L. Gissmann, and H. zur Hausen, 1981, J. Virol. 40, 932-935). However, sequence analysis of the upstream regulatory region identified several alterations in the purine-thymidine-rich region spanning nucleotides 7292-7400. One insertion of 24 bp at position 7323 represented an exact tandem duplication of nucleotides 7300-7323. A second insertion of 58 bp at position 7350 had 84% positional identity to immediately adjacent HPV 6 sequences (nucleotides 7303-7356) and was also similar to the 74-bp insertion found at position 7348 in HPV-6vc, cloned from an invasive verrucous carcinoma of the vulva (R. F. Rando, D. E. Groff, J. G. Chirikjian, and W. D. Lancaster, 1986, J. Virol. 57, 353-356). A deletion of 49 bp (nucleotides 7351-7399) immediately followed the 58-bp insertion. The cloned HPV6-T70 DNA induced the morphological transformation of NIH 3T3 cells.