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 protein 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 50% of these amino acids are identical between groups I and II. 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 group-specific polymerase chain reaction was performed within the genome region corresponding to the putative NS5 protein, where the group II hepatitis C virus genome is 57 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 5' untranslated region and in 78 patients by this group-specific 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 group-specific 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%).(ABSTRACT TRUNCATED AT 250 WORDS)