A case control study was conducted to identify the association of therapeutic injections with acute hepatitis B virus (HBV) infection in Karachi, Pakistan. We enrolled 67 cases of acute HBV infection (IgM anti-HBc positive) and 247 controls (anti-HBc negative) from four hospitals of Karachi during July 2000-June 2001. Exposure to various risk factors during the period relevant to the incubation period of HBV was recorded both from cases and controls using a structured questionnaire. Multivariate logistic regression analysis of the data showed that cases were more likely to have received one injection (OR = 4.0; 95% CI 1.4, 11.1), or more than one injection (OR= 6.3; 95% CI 3.2, 12.4) compared to controls. The estimated population attributable risk (PAR) for therapeutic injections was 53%. Also the cases compared to controls were more likely to have household size of seven or more (OR = 1.9; 95% CI 0.95, 3.9). This study showed that unsafe therapeutic injections appear to be the major risk factor for acute HBV infection and needs immediate focus from public health stand point.