This study was conducted to evaluate reports that hepatitis B virus (HBV) DNA sequences can be found in the serum and/or tumour tissue from some hepatocellular carcinoma (HCC) patients who have no detectable hepatitis B surface antigen (HBsAg) in their sera. Such HBV infections would be highly atypical, because prospective studies have shown a clear succession of specific serologic markers during and after most HBV infections. As most HBsAg-negative HCC patients in Japan have hepatitis C virus (HCV) infections, the present study was conducted to determine whether some of these patients actually have unrecognized HBV infections. Thirty newly diagnosed HCC patients from Kurume, Japan, with antibody to the hepatitis C virus (anti-HCV) were studied. None of the 30 had HBsAg detectable in their serum. Of 22 for whom test results for antibodies to the hepatitis B core antigen (anti-HBc) and antibodies to HBsAg (anti-HBs) were available, 14 (64%) had anti-HBc and anti-HBs, four (18%) had anti-HBc alone, and four (18%) had no HBV markers. Nested polymerase chain reaction was used to detect the HBV surface (S), core (C), polymerase (P) and core promoter gene sequences in the HCC tissues and in the adjacent nontumorous liver tissues. HBV DNA was detected in HCC and/or adjacent nontumorous liver in 22 of 30 (73%) patients [detected in both HCC and nontumorous liver in 19/30 patients (63%)]. Among the 22 patients with detectable HBV DNA, more than one HBV gene was detected in 10 (46%). Among the four patients whose sera were negative for all HBV markers, three had HBV DNA in either HCC and nontumorous liver (two cases) or only in the nontumorous liver (one case); HBV DNA could not be detected in tissues from the fourth patient. In 18 of 21 (86%) patients with detectable HBV core promoter sequences, mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found. No deletions were detected in the core promoter gene region of the type reported to be associated with some cases of HBsAg-negative HBV infection. Thus, HBV DNA was detectable in 22 (73%) HBsAg-negative, anti-HCV-positive HCCs, including three (10%) who were also negative for anti-HBc and anti-HBs. HBV mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found in the majority of cases, mutations that have previously been reported in HBV that is integrated in HCC DNA. In serologic surveys to determine etiologic associations of HCC, patients such as those in this study would have been incorrectly designated as having 'HCV-associated HCC,' whereas the data in this study suggest that HBV could have played a role in the development of their HCCs.