BACKGROUND The prevalence of HIV/hepatitis B virus (HBV) co-infection in South Africa ranges from 4.8% to 17% using the standard marker surface antigen (hepatitis B surface antigen, HBsAg) for chronic active HBV infection. However, sensitive molecular techniques for detecting HBV DNA in serum can detect occult HBV infection. We report the first observational prospective study of occult HBV infection in HIV-positive people in South Africa. METHODS Five hundred and two patients attending an urban hospital were screened for HBV using serological testing for HBsAg, core antibody (anti-HBc), and surface antibody (anti-HBs). DNA was analyzed using real-time quantitative PCR to determine the HBV viral load. RESULTS Of the 502 participants, 24 (4.8%) were HBsAg-positive and 53 (10.6%) were positive for anti-HBc alone. Of these 53, screening for occult disease was carried out in 43, of whom 38 (88.4%) were positive. The mean HBV viral load was 2.8 x 10(4) copies/ml (range 1 x 10(2) to 1 x 10(6) copies/ml). CONCLUSIONS Combining the participants with positive HBsAg and occult HBV DNA results, the prevalence of HBV increases from 4.8% (HBsAg alone) to 12.4%. While the clinical impact of occult HBV infection is unclear, consideration should be given to changing the guidelines to recommend dual HBV therapy for the treatment of co-infected patients in the developing world.