Taiwan has been facing a rising epidemic of human immunodeficiency virus (HIV) infection since 2004. Injection drug users comprised 38.5% of accumulated HIV cases by 2007. This cross-sectional study investigated the seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and HIV infection in 753 male substance users who were detained in a detoxification center in Taoyuan, Taiwan. The subjects were enrolled into the study consecutively between February and October, 2005. The seroprevalence rates of HIV antibodies, HCV antibodies, and HBV surface antigens among all subjects, and HDV antibodies among HBV carriers were 6.9% (95% confidence interval [CI]: 5.19–8.95), 30.5% (95% CI: 27.23–33.93), 16.9% (95% CI: 14.24–19.71) and 13.7% (95% CI: 8.19–21.04), respectively. Subjects in the heroin injection group had significantly higher rates of HIV infection, HCV infection and HDV superinfection (25.5%, 89.6%, and 38.7%) than those in the heroin non-injection group (0.9%, 24.5%, and 6.25%), the methamphetamine group (0.3%, 8.1%, and 6.7%), and the club drug group (1%, 3%, and 0%; P < 0.001). The odds of HCV, HIV, or HDV infection were 74.7, 63.8, and 11.1 higher, respectively, for heroin injection drug users than for non-injection drug users (P < 0.0001). Compared to HIV-negative individuals, the odds of being a heroin injector and the odds of HCV co-infections were 64-fold and 149-fold higher, respectively, in HIV-positive individuals. The impact of HBV, HCV, and HDV infection on the HIV epidemic in Taiwan should be monitored closely. J. Med. Virol. 81:973–978, 2009. © 2009 Wiley-Liss, Inc.