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.