A recently discovered non-A-E hepatitis virus has been designated as hepatitis G virus (HGV) and identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood-borne hepatitis and usually in the presence of hepatitis C or hepatitis B virus (HBV) infection. In this study, the presence of HGV-RNA in serum or plasma and the prevalence of antibodies against an HGV envelope protein (E2) were investigated in patients undergoing chronic hemodialysis using a sensitive reverse-transcriptase polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. HGV-RNA was detected in 19 of 112 patients investigated (17%) and anti-E2 antibodies were detected in 15 of 106 patients studied (14.2%). With the exception of two patients, the appearance of anti-E2 is associated with the clearance of serum HGV-RNA. The total prevalence of current (HGV-RNA positivity) and/or past (anti-E2 positivity) HGV infection in this patient population is thus 28.6% (32 of 112 patients were positive for serum HGV-RNA and/or anti-E2 antibodies). In apparently healthy blood donors, serum HGV-RNA was detected in four of 358 individuals (1.12%) and anti-E2 was not detected in 50 individuals investigated. From the 19 patients with serum HGV-RNA positivity, nine were coinfected with other hepatitis viruses (seven with HBV; one with HBV, hepatitis C virus [HCV], and hepatitis D virus; and one with HBV and cytomegalovirus). Thirteen of 15 patients with anti-E2 positivity (10 were positive for only anti-E2 and three were also positive for anti-HBc) had no detectable HGV-RNA. In two patients, both HGV-RNA and anti-E2 antibodies were concomitantly present (both patients were coinfected with HCV or HBV). Of the HGV-infected patients, only three who were coinfected with HBV showed elevated serum alanine aminotransferase levels. The serum HCV-RNA and/or anti-HCV were detected in five (4.5%) of 112 patients. From these findings, we conclude that there is a high prevalence of HGV infection (28.6%) compared with HCV (4.5%) in patients undergoing hemodialysis in our hospital. However, approximately 50% of patients had spontaneously lost the viremia and developed anti-HGV-E2 antibodies. We confirm that HGV infection alone is not associated with elevated serum transaminases, and the appearance of anti-HGV-E2 is usually accompanied with clearance of serum HGV-RNA. In contrast to the results of our previous study, the majority of patients infected with HGV are not coinfected with HCV, indicating that HGV is capable of independent transmission. It is likely that there is a preferential HGV acquisition in the hemodialysis unit. The clinical significance of long-term infection with HGV remains to be established.