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To determine the risk of developing high grade anal squamous intraepithelial neoplasia (HG-AIN) in relation to HIV infection, and immunosuppression, after controlling for the effects of human papillomavirus (HPV) infection.Prospective cohort study of 158 HIV-seropositive, and 147 HIV-seronegative homosexual men presenting to a community-based clinic with initially negative anal cytologic, and colposcopic findings.Subjects completed self-administered questionnaires, underwent cytologic screening, and standardized unaided, and colposcopic examination of the proximal anal canal for presence of abnormalities suggestive of AIM. Anal specimens were screened for HPV DMA.HG-AIN developed in eight (5.4%), and 24 (15.2%) HIV-seronegative, and -seropositive men, respectively. Risk of HG-AIN among HIV-seronegative men was associated with detection of anal HPV types 16 or 18 by Southern transfer hybridization (STH), detection of HPV 16 or 18 at the lower levels by polymerase chain reaction but not by STH, and with number of positive HPV tests; HG-AIN risk among HIV-seropositive men was associated with detection of HPV 16 or 18 only by STH, detection of HPV types other than 16 or 18, CD4 count ≤ 500 × 106/l, and number of positive HPV tests. HIV-induced immunosuppression remained an independent predictor of HG-AIN after adjusting for type, and level of detection of HPV; HIV infection predicted HG-AIN risk after adjustment for number of positive HPV tests.The association of HG-AIN with HIV, independent of HPV type, level of HPV detection, and number of positive HPV tests, suggests that this increased risk cannot be entirely explained by an effect of HIV on HPV detection. Future studies focusing on factors more specific to the local microenvironment in the anal canal should help clarify these issues.