Summary Background A flavivirus designated hepatitis G virus (HGV) has been isolated from the serum of patients with non-A–E hepatitis. Hitherto, the presence of HGV RNA in serum has been detected with the reverse transcription-polymerase chain reaction (RT-PCR) amplification method. We have now developed an immunoassay for antibodies against an HGV protein. Methods Recombinant HGV envelope protein E2 was used as antigen in an ELISA. 80 blood donors, 99 intravenous-drug users, and 11 patients with acute post-transfusion hepatitis were tested for antibodies to E2. The HGV-RNA status was assessed by RT-PCR. Findings Anti-E2 seroprevalence was 9% among the blood donors and 41% among the drug users; HGV-RNA prevalence was 2·5% and 38%, respectively. Whereas anti-E2 prevalence increased with the duration of drug use, HGV-RNA prevalence declined in parallel. In each group, the presence of anti-E2 and HGV RNA was almost mutually exclusive: none of the blood donors and only 4% of the drug users were positive for both markers at the same time. Of the 11 post-transfusion patients—who were all HGV-RNA positive and anti-E2 negative at the onset of disease—four developed antibodies to E2 during the following year, and two of the four subsequently became HGV-RNA negative. Interpretation We conclude that a humoral immune response to E2 is associated with loss of detectable HGV viraemia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. The immunoassay we describe should facilitate investigation of suspected infections and may be helpful in the elucidation of the clinical significance of HGV.