Objectives: To estimate the prevalence and incidence of hepatitis B virus (HBV) infection among patients attending three STD clinics in Pune, India, and to identify associated risk factors. Methods: Of the 2098 patients screened at STD clinics in Pune during 1996, 497, who returned for at least one follow up visit, were screened for various markers of HBV infection (HBsAg, anti-HBs, anti-HBc), HIV antibody, and VDRL. Results: Of the 497 participants 3.6%, 26.5%, and 43.2% were positive for HBsAg, anti-HBs, and anti-HBc respectively. Tattooing (AOR 1.64, 95% Cl 1.03 to 2.64) was found to be independently associated with presence of core antibody. Additionally, history of being in commercial sex work and history of a genital ulcer were independently associated with a positive anti-HBc antibody test (AOR 12.45, 95% Cl 5.58 to 27.82 and AOR 1.70, 95% Cl 1.09 to 2.66, respectively). 72 out of 497 (14.5%) participants were HIV positive at baseline. HIV-1 antibody positive patients were more likely to have a positive anti-HBc test (69.4% v 39.0%, p<0.001). 30 out of 282 participants, negative for anti-HBc antibody at enrolment, seroconverted subsequently, resulting in an incidence of 10.86 per 100 person years (95% Cl 7.2%, 14.5%) (mean and accumulated follow up of 11.7 months and 276.17 person years, respectively). Conclusions: A high prevalence and incidence of HBV infection, seen in STD clinic attendees underscore the need to provide HBV vaccine to commercial sex workers and their clients in India.