Polymerase chain reaction (PCR) and in situ hybridization were used to test for the presence of human papillomavirus (HPV) DNA in cases of anorectal squamous cell carcinoma. Human papillomavirus was detected by PCR with L1 consensus sequence primers in 22 of 27 cases, including 10 of 11 cases with a prominent basaloid pattern and 12 of 16 cases without basaloid patterns of differentiation. Slot blot hybridization identified HPV type 16 as the most common type, present in 7 of 10 cases of basaloid carcinoma and 10 of 12 cases without basaloid features. In situ hybridization confirmed the presence of HPV in tumor cell nuclei of five cases of basaloid carcinoma and in eight cases of squamous cell carcinoma without basaloid pattern. The authors conclude that the prevalence of HPV in cases of anorectal squamous cell carcinoma is unrelated to the presence or absence of a basaloid pattern of differentiation.