The objective of this study was to determine the frequency and load of hepatitis B virus (HBV) DNA in anti-HBc-positive first-time blood donors; it was designed to contribute to determining whether anti-HBc screening of blood donations might reduce the residual risk of posftransfusion HBV infection. A total of 14 251 first-time blood donors were tested for anti-HBc using a microparticle enzyme immunoassay; positive results were confirmed by a second enzyme-linked immunosorbent assay (ELISA). For the detection of HBV DNA from plasma samples, we developed a novel and highly sensitive real-time polymerase chain reaction (PCR) assay. The 95% detection limit of the method amounted to 27.8 IU/mL, consistent with the World Health Organization (WHO) international standard for HBV DNA. A total of 216 blood donors (1.52%) tested anti-HBc-positive in both tests, and 205 of them (16 HBsAg(+), 189 HBsAg(-)) were tested for HBV DNA. In 14 (87.5%) of the HBsAg-positive blood donors, HBV DNA was repeatedly detected, and in 3 (1.59%) of the HBsAg-negative donors, HBV DNA was also found repeatedly. In the 3 HBV DNA-positive, HBsAg-negative cases, anti-HBe and anti-HBs (> 100 IU/L) were also detectable. HBV DNA in HBsAgnegative as well as HBsAg-positIve samples was seen at a low level. Thus, HBV DNA is sometimes found in HBsAgnegative, anti-HBc-positive, and antiHBs-positive donors. Retrospective studies on regular blood donors and recipients are necessary to determine the infection rate due to those donations. Routine antiHBc screening of blood donations could probably prevent some transfusion-transmitted HBV infections. (C) 2002 by The American Society of Hematology.