To investigate the association between GB virus C/hepatitis G virus (GBV-C/HGV) infection and the development of hepatocellular carcinoma (HCC) in H city, in the inshore area of the Yangtze River, where high prevalence of HCC has been reported, we determined hepatitis B virus (HBV) and hepatitis C virus (HCV) markers, GBV-C/HGV-RNA and GBV-C/HGV E2 antibody (anti-HG E2) among 114 HCC patients and the same number of age- and sex-matched controls. There were no significant differences in the clinical and demographic characteristics between them, except for serum alanine aminotransferase level and history of liver diseases. There was a significant difference of hepatitis B virus surface antigen (HBsAg) prevalence between the HCC patients (75.4%) and the controls (20.2%; P<0.01). Hepatitis C virus antibody was detected in 4.4% of the HCC patients, compared with 1.7% of the controls. GB virus-C/HGV-RNA and anti-HG E2 were detected in 14.9 and 1.7% of the HCC patients, respectively, compared with 7.0 and 1.7% of the controls, respectively. Nucleotide sequences and molecular evolutionary analysis showed the strains of GBV-C/HGV-RNA were classified into genotype 2 and 3 (HG and ASIA type). An effect analysis showed an odds ratio (OR) for developing HCC from GBV-C/HGV infection among HBsAg-positive subjects was 14.9, with a 95% CI of 4.9-45.4. HBsAg infection alone was 13.83 (95% CI 7.4-25.9) and GBV-C/HGV infection alone, 3.74 (95% CI 1.1-13.1), respectively. These data indicate that HBV infection is considered to be one of the major risk factors in patients with HCC and although GBV-C/HGV infection was observed in both the HCC and the control groups, it might not play an important role in the development of HCC in this area.