BACKGROUND: Screening of blood donations for antibodies against hepatitis B core antigen (anti-HBc) is an accepted method to prevent some transfusion-transmitted hepatitis B virus (HBV) infections. However, anti-HBc testing may result in donor loss due to unspecific results in the currently available anti-HBc tests. Algorithms to distinguish true-positive from false-positive results and for reentry of those donors who tested false anti-HBc positive were evaluated retrospectively. STUDY DESIGN AND METHODS: Samples that tested reactive for anti-HBc by chemiluminescent microparticle immunoassay (CMIA) were investigated for anti-HBc by microparticle immunoassay, for anti-HBs and hepatitis B surface antigen (HBsAg) by CMIA, and for HBV DNA by individual-donor nucleic acid testing. Results were classified true positive, indeterminate, and false positive for anti-HBc. Donors who tested indeterminate and false positive were admitted for reentry if follow-up testing for anti-HBc became negative and no further evidence for an HBV infection was apparent. RESULTS: A total of 554 of 148,000 samples, taken from 30,000 individuals within 3 years tested reactive for anti-HBc by CMIA. Of those, 553 could be further classified: 142 (26%) true positive, 76 (14%) indeterminate, and 335 (60%) false positive. A total of 214 of 411 (52%) samples termed indeterminate or false positive were admitted for reentry and able to provide further donations. In one donor, anti-HBc-positive/HBsAg- and HBV DNA-negative HBV DNA was detectable during follow-up. CONCLUSION: According to our proposed algorithm, 26% of anti-HBc-reactive results tested by CMIA were true positive. Many donors tested indeterminate or false positive can provide future donations if our proposed algorithm for reentry is applied. One donor at risk for transmitting HBV was identified solely by anti-HBc testing.