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Injection drug use (IDU) is one of the most significant risk factors for viral hepatitis (B, D and C) and human immunodeficiency virus (HIV) infection. However, there is little information about the risk of infection among non-injection drug users (non-IDUs). The present study was designed to perform several objectives: (a) to evaluate the prevalence of serological markers of hepatitis B, D, C virus and HIV in IDU and non-IDU patients; (b) to compare the prevalence of these markers between both groups; (c) to identify risk factors for HCV and HIV in this population; and (d) to correlate the presence of HCV and liver function. A total of 385 consecutive patients (122 IDUs and 263 non-IDUs), admitted to the Drug Dependency Treatment Unit at the Hospital Insular of Gran Canaria between 1993 to 1994, were included in the study. The serological markers of HBV, HDV, HCV and HIV were determined by ELISA and immunoblot methods. In all cases we also measured syphilis tests (RPR and FTAabs), serum aminotransferases and serum gammaglutamiltranspeptidase. Compared to the non-IDU, the IDU group presents a higher prevalence of antiHBc (55.0% vs. 20.7%, p < 0.0001), antiHCV (87.6% vs. 35.3%, p < 0.0001) and antiHIV (21.8% vs. 2.7%, p < 0.0001). There was no significant difference in RPR positivity (0.9% vs. 4.9%, p = 0.06). Delta infection was only detected in injection drug users, and the prevalence was low. Using logistic regression, the only risk factors associated with antiHCV positivity were injection drug addiction (OR: 9.2, 95% CI: 4.9–17.0) and antiHBc positivity (OR: 5.5, 95% CI: 3.0–9.9). Similarly, the associated risk factors for HIV were injection drug addiction (OR: 5.9, 95% CI: 2.3–15.0) and antiHBc positivity (OR: 3.8, 95% CI: 1.5–9.2). However, no correlation was found between antiHCV positive and antiHIV or between these markers and RPR positivity. Patients positive for antiHCV showed significant elevations in aspartate aminotransferase and alanine aminotransferase levels, when compared with patients negative for antiHCV: 65.0 vs. 39.2 U.l (p < 0.001) and 88.4 vs. 40.3 U/l (p < 0.001), respectively. We conclude that drug users have an elevated prevalence of HCV, HBV and HIV infection, even if drug use is only inhalated. On the other hand, the main risk factors associated with HCV and HIV are injection drug addiction and exposure to hepatitis B virus. Finally, in the study population, liver dysfunction is closely related to HCV infection.