Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of squamous cell carcinoma of the anorectal region. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In cervical and head and neck cancers, HPV infection is also associated with an overexpression of p16, a cyclin-dependent kinase inhibitor. The expression of these cell cycle regulators in squamous cell carcinomas of the anorectal region has not been well studied. In the current study, 29 cases of squamous cell carcinoma of the anorectal region were immunohistochemically examined for the expression of p16, Rb, and p53 proteins. Tumor cell DNA was also extracted from paraffin blocks and subjected to broad-spectrum HPV DNA testing and typing. The results show that the tumor cells exhibited a strong and diffuse nuclear stain (with some cytoplasmic positivity) for p16 in all 29 cases (100%). The adjacent nonneoplastic squamous epithelium or colonic mucosa, in contrast, was completely negative. Loss of Rb nuclear staining in tumor cells was observed in 20 cases (69%). The p53 protein was essentially undetectable, with only 6 cases containing <10% positive cells. HPV DNA was detected in every case (100%), with 25 cases (86%) harboring Type 16. In addition, almost identical results were obtained in 12 HPV-positive squamous cell carcinomas of the upper aerodigestive tract. This was in marked contrast to those of HPV-negative tumors, where positive p16 staining and loss of Rb expression were seen in only 2/21 (10%) and 1/21 (5%) cases, respectively. These observations indicate that overexpression of p16 and loss of Rb nuclear staining are commonly associated with high-risk HPV infection, which may serve as useful surrogate biomarkers for identifying squamous cell carcinomas harboring HPV DNA.