Because large numbers of volunteer blood donors may be disqualified for “false-positive” results on tests for antibody to hepatitis B core antigen (anti-HBc), a more specific definition of anti-HBc enzyme immunoassay (EIA)-reactive was evaluated, including only those donor samples that were “strongly” reactive (sample-to-cutoff absorbance ratio, < 0.45). Results using this definition and other anti-HBc test methods were compared to the serologic response (antibody to hepatitis B surface antigen [anti-HBsAg]) to hepatitis B vaccination. Fifty-eight volunteer blood donors who had previously been deferred as donors, because of reactive anti-HBc tests (all other blood screening tests were negative, including those for HBsAg and anti-HBsAg) on two occasions, were vaccinated for hepatitis B. It was assumed that an anamnestic response to vaccine indicated past infection with hepatitis B, while a primary response to vaccine indicated lack of past infection. One (2%) of 43 donors with a historically “weak” anti-HBc (reactive absorbance ratio, > or = 0.45) had an anamnestic response to vaccine, compared to 8 (53%) of 15 with historically “strong” anti-HBc (reactive absorbance ratio, < 0.45) (p < 0.005). Anti-HBc testing using the microparticle EIA method also correlated well with hepatitis B vaccination results. The use of a narrower definition of “reactive” for anti-HBc EIA testing yielded much more specific, but slightly less sensitive, results.