PURPOSE:The purpose of this study was to determine the incidence of human papillomavirus deoxyribonucleic acid (HPV DNA) in anal squamous carcinoma.METHODS:HPV DNAin situhybridization for HPV Types 6, 11, 16, 18, 31, 33, and 35 was performed on the formalin‐fixed, paraffinembedded tissue from 53 perianal and anal squamous carcinomas and 10 controls.RESULTS:HPV DNA sequences were identified in 18 of 53 anal squamous carcinomas (34 percent). All 10 controls were negative for HPV DNA. Of the 18 positive patients, 10 were perianal squamous carcinomas, and 8 were anal canal squamous carcinomas. Six of the perianal carcinomas were positive for HPV Types 6 and 11. The remaining four perianal carcinomas and all eight of the anal canal carcinomas were positive for HPV Types 16 and 18.CONCLUSION:HPV DNA sequences can be identified in anal squamous carcinomas. Anal squamous epithelium is another site where HPV infection may carry a risk for malignant transformation. One‐third of anal squamous carcinomas may be associated with prior HPV infection. Patients with anogenital HPV infection should be routinely screened for anal squamous lesions.