Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of primary small cell carcinoma of the uterine cervix, a rare but highly aggressive malignancy. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In squamous cell carcinoma and adenocarcinoma of the cervix, HPV infection is also associated with overexpression of p16, a cyclin-dependent kinase inhibitor. In this study, 22 cases of primary small cell carcinoma of the uterine cervix were subjected to broad-spectrum HPV DNA amplification and typing, and immunohistochemically examined for the expression of p16, Rb, and p53 proteins. The results show that HPV DNA was detected in every case (100%), with 18 cases (82%) harboring type 18. The tumor cells exhibited strong nuclear staining for p16 in 20 cases (91%). This was associated with a complete loss of Rb nuclear staining in tumor cells in 16 cases (73%). The p53 protein was essentially undetectable in all cases. In contrast, HPV DNA was not detected in 9 colorectal and 8 urinary bladder small cell carcinomas included in this study for comparison. While similar p16 and Rb expression patterns were observed in these HPV-negative tumors, a different expression pattern for p53 was noted where strong nuclear staining was seen in 8 cases (47%; P = 0.0004 compared with cervical tumors). These observations indicate that different mechanisms are involved in the pathogenesis of small cell carcinomas of the uterine cervix and support the notion that nuclear p16 overexpression serves as an indication of Rb defunctioning in tumor cells, which may or may not result from high-risk HPV infection.