A new hepatitis-associated RNA virus of the Flaviviridae family has been identified and named GB virus C/ hepatitis G virus (HGV). We carried out a case-control study to evaluate the association of HGV infection with hepatocellular carcinoma (HCC). We recruited 170 patients hospitalized for HCC (143 male and 27 female, mean age 64 years) and 306 patients hospitalized for nonliver diseases (controls) in Brescia, Italy. HGV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and antibodies against HGV E2 protein (anti-E2) by an immunoassay test. HGV RNA was found in 8 cases (4.7%) and 4 controls (1.3%). The relative risk (RR) for HGV RNA positivity adjusted for demographic variables and hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) RNA, and alcohol was 7.3 (95% confidence interval, 1.7-30.6; P = .009). No HGV RNA-positive subject was also positive for anti-E2. Anti-E2 prevalence did not differ significantly between cases (20%) and controls (15.3%), and no RR increase was found by this marker. Among subjects with HGV exposure (HGV RNA plus anti-E2 positive), a greater proportion of cases (40%) than controls (14%) had transfusion history. The possible role of HGV in HCC etiology seems modest because the population-attributable risk is lower (4%) than those for HBsAg (22%), HCV RNA (36%), and heavy alcohol intake (52%). This study supports the hypothesis of an association between HGV infection and HCC, although at present there are insufficient data on the causality of the association.