Sequences obtained in the 5′ non-coding region (5′NCR) of hepatitis C virus (HCV) were obtained from Scottish blood donors and compared with previously published HCV sequences. Phylogenetic analysis revealed the existence of three distinct groups of sequences; two of these corresponded to the recently described HCV types 1 and 2 variants, while viral sequences detected in around a third of the blood donors formed a separate phylogenetic group that probably represents infection with a novel virus species. Nucleotide sequences of this latter group differed from all previously published 5′NCR sequence variants by at least 9%. This new virus type also differed considerably from previously published variants in other regions of the viral genome (core, NS-3 and NS-5), with corrected nucleotide distances of 15, 43 and 49% respectively from the prototype HCV-1 sequence. Formal phylogenetic analysis of each of the coding regions confirmed that HCV type 1 variants could be clearly differentiated into regional variants (Far East and U.S.A./European), in contrast to the clearly overlapping geographical distributions of the main HCV types in U.K. blood donors. We discuss the evidence for and against the hypothesis that the three main phylogenetic groups identified in this study represent separate species of HCV.