Infection with human papillomavirus (HPV), especially HPV16, is central to the development of squamous anogenital cancers and their precursor lesions, termed "squamous intraepithelial neoplasias." Men who have sex with men, particularly those who are infected with HIV, are at a high risk for anal infection with HPV16 and for low-grade anal neoplasia; however, only a subset of these men develop anal invasive cancer or its immediate precursor lesion, anal carcinoma in situ (CIS). To examine the hypothesis that certain variants of HPV16 are most strongly associated with development of anal CIS, we followed 589 men who have sex with men whose initial anal cytological smears did not show anal CIS. Anoscopy, anal cytology, and PCR-based assays for detection and classification of HPV types were performed every 4-6 months, with HPV16 further classified by single-stranded conformation polymorphism analysis as being a prototype-like (PL) or non-prototype-like (NPL) variant. Anal CIS was histologically confirmed in 6 of 384 (1.6%) consistently HPV16-negative men, in 12 of 183 (6.6%) men with HPV16 PL variants, and in 4 of 22 (18.2%) men with HPV16 NPL variants. After adjustment for anal cytological diagnoses at study entry, HIV status and CD4 count, and detection of HPV types other than type 16, men with HPV16 NPL variants were 3.2 times (95% confidence interval, 1.0-10.3) more likely to develop anal CIS than were those with PL variants. Neither detection of HPV16 DNA at high levels nor detection of HPV16 DNA for a prolonged period, factors that we previously demonstrated to be associated with risk of high-grade anal squamous intraepithelial neoplasia, was significantly associated with HPV16 NPL variants. The biological mechanism relating to Ihis excess risk remains undetermined.