Some individuals have “occult” infection with hepatitis B virus (HBV), defined as presence of HBV genome in the serum or liver tissue without HBV surface antigen (HBsAg) in the serum. The aim of this study was to investigate whether serum antibodies against HBV core antigen in isolation (“anti-HBc alone”) are a useful marker of “occult” HBV in patients with or without hepatitis C virus (HCV) infection. “Anti-HBc alone” was detected in the sera of 119/6,544 (1.8%) asymptomatic outpatients referred to the diagnostic laboratory for routine testing for viral hepatitis, 62/607 (10.2%) drug users, and 42/195 (21.5%) patients with hepatocellular carcinoma. Using three in-house nested-PCR amplification assays to detect HBV preS-S (S), precore-core (C), and Pol viral regions, respectively, “occult” HBV sequences were found in 9 of the 223 sera (4.0%) with “anti-HBc alone.” The highest prevalence of “occult” HBV sequences (5.9%) was detected in “anti-HBV alone” sera of individuals referred to the diagnostic laboratory without HCV antibodies. Direct sequencing of all PCR products confirmed the specificity of the PCR reactions and revealed the predominance of HBV genotype D. The data presented in this study suggest that detection of “anti-HBc alone” could reflect unrecognized “occult” HBV infection and that physicians should consider investigating such patients with HBV molecular tests. J. Med. Virol. 80:577–582, 2008. © 2008 Wiley-Liss, Inc.