Epidemiologic and clinical evidence has suggested a possible association between anal cancer and human papillomavirus (HPV) types that are known to be associated with cervical and other genital cancers. Using Southern blot and dot blot analysis, the authors examined 45 primary anal malignancies for HPV DNA types 6, 11, 16, and 18. HVP 16, DNA was detected in 23 of 41 (56 percent) anal squamous-cell carcinomas (SCC) and in the lymph-node metastases of two of these tumors. In addition, HPV 18 DNA was detected in 2/41 (5 percent) anal SCCs. Anal SCC contained no detectable HPV 6 or 11 DNA. The remaining four primary anal malignancies were not squamous carcinomas and did not contain any detectable HPV DNA. Nonmalignant anal epithelium and malignant rectal mucosa obtained from surgical patients undergoing hemorrhoidectomy and abdominoperineal excision of the rectum did not contain any detectable HPV DNA. HPV 16 DNA in anal cancer was predominantly integrated into the host cell DNA. In situ hybridization was used to demonstrate that HPV 16 DNA in anal SCC tissues is confined to the nuclei of carcinoma cells. The results of this investigation closely parallel similar studies of cervical cancer and lend support to the concept of the involvement of HPV 16 and 18 in the development of anal and genital squamous-cell carcinoma.