To determine if heterosexual activity was a risk factor for acquiring hepatitis B virus (HBV) infection, we studied attendees at a sexually transmitted disease (STD) clinic and students at a large university. Responses to self-administered questionnaires were used to exclude persons with factors known to be related to the acquisition of HBV and to determine the number of recent (past four months) and lifetime sexual partners. Serum samples were tested for hepatitis B surface antigen, antibody to hepatitis B surface antigen, and antibody to hepatitis B core antigen. For white heterosexuals in the STD group, the prevalence of HBV infection was 6% for those with fewer than five recent partners vs 21% for those with five or more partners, and 5% for those with fewer than 50 lifetime partners vs 20% for those with 50 or more partners. For black heterosexuals in the STD group, no association was found between prevalence of HBV infection and number of recent or lifetime sexual partners. For the heterosexual student group (all white), the prevalence of HBV infection was 1.5% for fewer than three recent partners vs 14% for three or more partners, and 2% for fewer than ten lifetime partners vs 7% for ten or more partners. For white heterosexuals, the risk of acquiring HBV infection increases with increasing amounts of sexual activity and may reach a level similar to that of other groups previously recognized to be at high risk of acquiring hepatitis B.