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Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMerieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.