This study assessed whether behavioral differences explained higher human immunodeficiency virus (HIV) seroprevalence among injection drug users (IDUs) in three East Coast versus two West Coast cities in the United States. Sociodemographic, sexual, and injecting information were collected during semiannual face-to-face interviews. Baseline data from New York City; Baltimore, Maryland; and New Haven, Connecticut, were compared with data from Los Angeles, California, and San Jose, California. Among 1,528 East Coast and 1,149 West Coast participants, HIV sero-prevalence was 21.5% and 2.3%, respectively (odds ratio [OR] 11.9; 95% confidence interval [CI] 7.9-17.8). HIV risk behaviors were common among IDUs on both coasts, and several were more common among West Coast participants. Adjusting for potential risk factors, East (vs. West) Coast of residence remained highly associated with HIV status (adjusted OR 12.14; 95% CI 7.36-20.00). Differences in HIV sero-prevalence between East and West Coast cities did not reflect self-reported injection or sexual risk behavior differences. This suggests that other factors must be considered, such as the probability of having HIV-infected injection or sexual partners. Prevention efforts are needed on the West Coast to decrease HIV-associated risk behaviors among IDUs, and further efforts are also needed to reduce HIV incidence on the East Coast.