The 5' end of the NS-4 protein of different genotypes of hepatitis C virus (HCV) is highly variable in nucleotide and inferred amino acid sequence, with frequent predicted amino acid substitutions between all six of the major HCV genotypes described to date. This region has been shown to be antigenic by epitope mapping, and elicits antibody in HCV-infected individuals with a detectable type-specific component. We have used this sequence data to specify branched peptides for an indirect binding/competition assay to detect type-specific antibody to each major genotype. A total of 183 out of 210 samples (87%) from blood donors and patients with chronic hepatitis C infected with genotypes 1 to 6 showed detectable type-specific antibody to NS-4 peptides that in almost all cases (> 97 %) corresponded to the genotype detected by a PCR typing method. These findings demonstrate the existence of major antigenic differences between genotypes of HCV, and indicate how infection with different variants of HCV may be detected by a serological test.