Objective To determine whether frontloading (i.e., syringe-mediated drug-sharing) is a risk factor for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection among injecting drug users (IDU). Design Cross-sectional study. Data on sociodemographic and behavioural characteristics were obtained by a standardized questionnaire. Serum samples were tested for seromarkers for HIV, HBV and HCV. Setting and participants IDU were recruited at 'low-threshold' storefront agencies (out-of-treatment sample), and at a centre for long-term drug use treatment (in-treatment sample). Individuals were included in the study if they had injected drugs within the previous 3 months. Main outcome measures Serological evidence for HIV, HBV, HCV exposure. Results Of all IDU (n = 324), 84% had ever practised frontloading with non-sterile injecting equipment, and 46% had done so more than 100 times; 32% had front-loaded during the 6 months prior to the interview. The crude seroprevalence rates for HIV, HBV and HCV increased with the overall frequency of frontloading, and reached 22, 71 and 94%, respectively, among IDU who had frontloaded more than 100 times. After controlling for confounding effects by logistic regression, having practised front-loading more than 100 times was significantly associated with HIV infection [adjusted prevalence odds ratio (POR) 3.5; 95% confidence interval (CI), 1.4–9], and HCV infection (adjusted POR, 5.4; 95% CI, 2.3–12), but not with HBV infection. Another independent risk factor for all three virus infections was needle-sharing in prison. Conclusions In communities where sterile injection equipment is readily available, and IDU have substantially reduced their overall levels of needle-sharing, the practice of frontloading appears to be a major risk factor for infections by blood-borne viruses among IDU. Prevention activities should specifically address this risk behaviour.