the clinical significance of isolated anti-hbc is still a challenge. to elucidate the real importance of this finding in our blood donors, an investigation algorithm was tested. one hundred and twelve isolated anti-hbc seropositive blood donors underwent clinical evaluation and retesting of hbv markers. those who presented repeatedly reactive isolated anti-hbc, received a single dose of hepatitis b recombinant vaccine to verify anti-hbs early response. a hbv-dna determination by pcr was done for those who did not test positive to anti-hbs after vaccine. the level of anti-hbc was recorded as a ratio of the sample-to-cut-off values (s:c ratio) in 57 candidates at donation. comparing true and false-positive anti-hbc results, the different s:c ratios of them were statistically significant and when less than 2, implying in a false-positive result probability over 80%. a high percent of false-positive results (16.07%) was verified after anti-hbc retesting. hbv immunity was characterized in 49.11%, either by anti-hbs detection in retesting (15.18%), or after a single dose hbv vaccination (33.93%). hbv-dna was negative in all tested donors. in conclusion, this algorithm was useful to clarify the meaning of isolated anti-hbc in most of our blood donors.