Because the sensitivities of individual hybridization techniques differ considerably, their role in accounting for the published frequencies of human papillomavirus (HPV) DNA in anal squamous cell carcinomas, ranging from 0 to 61%, must be investigated. With the use of biotinylated probes to HPV 6, 11, 16, 18, and 33, three hybridization techniques were performed on the same paraffin-embedded tissue blocks selected from 13 cases of anal squamous cell carcinoma. HPV DNA was detected in 0%, 62%, and 85% of cases with the use of in situ hybridization with horseradish peroxidase, in situ hybridization with alkaline phosphatase, and dot blot hybridization, respectively. By dot blot hybridization, 69% had HPV 16/6 and 15% had HPV 6/11. An HPV DNA frequency range of 0-85% in the same group of tumors with the use of three hybridization techniques indicates the influential role of the method on HPV DNA prevalences. HPV DNA was identified regardless of patient gender or type of squamous cell carcinoma. The presence of HPV 16 in 82% of the positive cases in supportive evidence of the carcinogenic role of the HPV in anal squamous cell carcinoma.