Group I and II hepatitis C virus genotypes were determined by a newly developed serological genotyping assay. This assay detected antibodies against group-specific recombinant proteins in the putative NS4 pro*in region (amino acid no. 1676-1760) by an enzyme-linked immunosorbent assay. This region of the hepatitis C virus peptide has many group-specific amino acids; fewer than 60% of these amino acids are identical between groups I and 11. Genotypes determined by the serological genotyping assay were compared with those determined by a method in which the polymerase chain reaction was used in 91 chronic hepatitis patients. The groupspecific polymerase chain reaction was performed within the genome region correeponding to the putative NS6 protein, where the group I1 hepatitis C virus genome is 67 nucleotides longer than that of group I. Among 91 chronic hepatitis C patients who had positive results in the second-generation hepatitis C virus antibody (core and NS3 region) assay, hepatitis C virus RNA was detected in 80 patients by polymerase chain reaction in the 6' untranslated region and in 78 patients by this group-8pcMc polymerase chain reaction. As a result, in 76 of 91 patients (84%) genotypes determined by the serological genotyping assay showed complete agreement with those determined by the groupspecific polymerase chain reaction, and none of the patients revealed a group opposite to that of hepatitis C virus genotype. The detection rate of the serological genotyping assay (89 of 91; 98%) was even higher than that of the polymerase chain reaction assay (78 of 91; 86%). Thus, serological genotyping assay is specific and sensitive for the determination of hepatitis C virus genotypes, and this enzyme-linked immunosorbent