Cross-sectional associations between human papillomavirus (HPV), anal squamous intraepithelial lesions (SIL), and human immunodeficiency virus (HIV) were studied in a cohort of gay men. HPV DNA was detected by generic and type-specific polymerase chain reaction (PCR) probes and hybrid capture assay (HC). HPV virus load was estimated by HC relative light unit (RLU) ratio. HPV prevalence, number of HPV types detected, and HC RLU ratios were each greater in HIV-positive than HIV-negative participants. Further, among HIV-positive men, HC RLU ratio was inversely associated with CD4 cell count. SIL was more frequent in HIV-positive participants, particularly those with a CD4 cell count <200/microL and was positively associated with HPV. Men with a high HC RLU ratio were nearly 3 times more likely to have SIL than were those both PCR- and HC-negative. These data support that HIV augments HPV-associated anal disease in this population.