Prevalence of the recently discovered GB virus C(GBV-C) was evaluated in a cohort of 49 Italian patients with acute or chronic hepatitis of unknown etiology (non-A-E hepatitis) and in a control group of 100 healthy blood donors. The GBV-C genomes could be detected by polymerase chain reaction (PCR) with reverse transcription in 35% of the acute and 39% of the chronic hepatitis patients; only 1 of the control subjects had a positive response. All PCR products hybridized with a specific probe in a colorimetric assay, and the analysis of the sequences of the amplified cDNAs fully confirmed the specificity of the assay. Furthermore, the alignment of the predicted translation products identified two recurrent amino acid substitutions in 6 patients, suggesting the possible existence of at least 2 different GBV-C subtypes. Thus, GBV-C may be an important agent, contributing, at least in Italy, to a significant number of the cases of hepatitis of unknown etiology.