Serum samples, which were found positive for anti-HBc and negative for HBsAg and anti-HBs during routine testing with Abbott enzyme immunoassays (EIA), were collected prospectively. The samples were obtained from patients with a high risk of hepatitis B. Further analysis was carried out using radioimmunoassays and tests for additional markers of hepatitis B. 84 of the 203 initially collected samples (41.4%) were found positive for anti-HBs by RIA. Of the 119 samples negative for anti-HBs by RIA, 103 were available for further investigation, and 35 (34.0%) of these were negative for anti-HBc by RIA. This indicates that low sensitivity of the anti-HBs EIA and non-specificity of the anti-HBc EIA may account for about 60% of the cases presenting with isolated anti-HBc at initial testing. Fifty-two samples positive for anti-HBc by RIA, with isolated anti-HBc confirmed after RIA testing for HBsAg and anti-HBs, were examined further: anti-HBe was detected in 16 sera, anti-HDV in one serum and HBV-DNA in 5 sera by PCR. Additional testing of serum samples with isolated anti-HBc is warranted.