OBJECTIVE The objective of this study was to evaluate the burden of sexual- and injection drug use-related infections in male prisoners in Karachi, Pakistan. METHODS We administered a structured questionnaire in a cross-sectional survey of 365 randomly selected imprisoned men. We analyzed blood for the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) by ELISA, and for syphilis by rapid plasma reagin with Treponema pallidum hemagglutination assay confirmation. Subjects with possible tuberculosis (World Health Organization criteria) provided sputum samples for an acid-fast bacillus smear and culture. RESULTS The prevalence of tuberculosis was 2.2% (95% CI 0.71-3.8%). Of 357 of the randomly selected prisoners (eight refused to give blood), 2.0% (95% CI 0.6-3.4) were HIV-infected; syphilis was confirmed in 8.9% (95% CI 6.0-11.8%), HBV in 5.9% (95% CI 3.5-8.3%), and HCV in 15.2% (95% CI 11.7-18.8). By self-report, 59.2% had used any illicit drugs, among whom 11.8% (95% CI 8.5-15.0) had injected drugs. The median length of stay in the prison had been 3.2 (range 1-72) months. CONCLUSIONS All four infections were prevalent among the prisoners in Pakistan. Prisons are excellent venues for infectious disease screening and intervention given the conditions of poverty and drug addiction. Collaboration with community-based health providers is vital for post-discharge planning.