An RT-PCR assay using primers from the 5'-UTR of the GBV-C/HGV genome was used to detect viremia, and a serological assay was used to detect past exposure to GBV-C/HGV, in sera from 106 imprisoned Greek intravenous drug users. High seroprevalence rates indicative of the parenteral route of transmission of the virus were found (32.1% for GBV-C RNA and 46.2% for anti-GBV-C E2). These rates were nonetheless lower in comparison to the corresponding rates of HCV infection markers (64.2% for HCV RNA and 77.4% for anti-HCV). Statistically significant univariate associations were observed between GBV-C-RNA positivity and younger age (P=0.006) and HCV-RNA positivity (P=0.024), as well as with higher serum alanine aminotransferase levels (P< 0.001); this latter association was shown to be independent of coinfection with HCV and of age by a multiple logistic regression model. Apparently, GBV-C/HGV had spread readily by needle-sharing in prison, while causing acute subclinical hepatitis in infected inmates. Phylogenetic analysis of the partial 5'-UTR of the GBV-C/HGV genome from 16 seropositive individuals, which delineated their grouping within genotype 2, also revealed a close genetic relationship between two sets of sequences from 4 drug addicts, 3 of whom admitted to sharing needles while imprisoned.