Synthetic peptides representing sequences encoded at the 5'-terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV-infected plasma donors. HCV sequence-specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence-specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380-436 was shown to contain at least one variant-specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5'-terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants.