Men who have sex with men (MSM) continue to account for the largest number of new HIV infections in the United States, but limited data exist on independent risk factors for infection beyond the early 1990s. The HIV Network for Prevention Trials Vaccine Preparedness Study enrolled 3257 MSM in 6 US cities from 1995 to 1997. HIV seroincidence was 1.55 per 100 person-years (95% confidence interval: 1.23-1.95) over 18 months of follow-up. On multi-variable analysis using time-dependent covariates, independent risk factors for HIV seroconversion were increased number of reported HIV-negative male sex partners (adjusted odds ratio (AOR) = 1.14 per partner, population attributable risk (PAR) = 28%), nitrite inhalant use (AOR = 2.2, PAR = 28%), unprotected receptive anal sex with an HIV unknown serostatus partner (AOR = 2.7, PAR = 15%) or HIV-positive partner (AOR = 3.4, PAR = 12%), protected receptive anal sex with an HIV-positive partner (AOR = 2.2, PAR = 11%), lack of circumcision (AOR = 2.0, PAR = 10%), and receptive oral sex to ejaculation with an HIV-positive partner (AOR = 3.8, PAR = 7%). Having a large number of male sex partners, nitrite inhalant use, and engaging in receptive anal sex explained the majority of infections in this cohort and should be targeted in prevention strategies for MSM.