Surgical specimens of 15 patients with early and 12 patients with advanced squamous cell carcinoma of the vulva and the penis were examined for the presence of human papillomavirus (HPV) type 6, 11, 16, and 18 DNA by Southern blotting (SB) and polymerase chain reaction (PCR) analysis. By SB, HPV type 16 DNA was detected in all early carcinomas and 2 of 12 cases of advanced squamous cell carcinoma (ISCC) of the vulva and penis. PCR revealed HPV DNA in four additional cases of vulvar and penile ISCC negative by SB. Three cases contained HPV16 and one HPV18. Two cases of vulvar and penile Buschke-Lowenstein (BL) tumor with malignancy and one case of vulvar verrucous carcinoma were also examined by both techniques. While BL tumors were associated with DNA of HPV 6 or 11, no HPV association was found for verrucous carcinoma. Our results confirm that the detection rate of HPV DNA in early vulvar and penile carcinomas is much higher than in invasive carcinomas. In addition, we have shown that in the lower genital tract, 50% of cases of ISCC are HPV16 correlated. The absence of HPV DNA (types 6, 11, 16, and 18) in the remaining 50% of cases of ISCC thus suggests that vulvar and penile ISCC may have more than one pathogenetic pathway.