Epidemiologic studies suggest that human papillomavirus (HPV) plays an important role in the development of squamous cell carcinoma of the anal canal. However, in previous studies the prevalence of HPV found in tumor varied from 0 to 85%. This study was performed to investigate the prevalence and physical state of HPV DNA in anal carcinomas. HPV infection was examined using in situ hybridization and polymerase chain reaction. HPV DNA was detected in 80/99 (81%) of the tumors, including 66/74 (89%) from females and 14/25 (56%) from males. HPV16 was demonstrated in 84% of the females and 52% of the males. HPV18, HPV31, and HPV33 could be demonstrated in 5% or less of the cases. In situ hybridization technique showed that 100% of the virus-infected cases had punctate signal and 67% had in addition a diffuse signal, indicating integrated and episomal virus DNA, respectively. The integrated and episomal HPV DNA coexisted in the majority of the tumor cell nuclei. This study indicates that infection and integration of HPV DNA may play an important role in the pathogenesis of anal canal carcinomas.