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Objectives: Few studies of hepatitis B virus (HBV) infection and hepatitis B vaccination have focused on women, and specifically, women who are at high risk. This study was designed to assess the extent of HBV infection and vaccination, level of knowledge about hepatitis B, motivators and barriers to accepting vaccination and uptake of hepatitis B vaccine. Design: From March 2005 to June 2006, 402 HIV-negative noninjection drug-using women at sexual risk were recruited, interviewed, and tested for markers of HBV infection. Results: Based on serologic testing, 16.7% were previously vaccinated against HBV, 31.1% were previously infected and 52.2% were still susceptible to HBV. Knowledge of HBV infection, transmission, and prevention was low with a mean of 6.1 of 12 knowledge items correctly identified as true or false; a substantial percent of women were not sure of the correct answer. Of the women still susceptible, 69.0% started the hepatitis B vaccine series after counseling given through the study. Conclusion: This study illustrates that there continues to be gaps in current strategies for administering hepatitis B vaccine among female populations at sexual risk. Interventions are needed for this population to increase awareness and knowledge of hepatitis B, its transmission, impact on health and the availability of a safe and effective vaccine, supplemented by community programs for adult vaccination.