Hepatitis C virus (HCV) genotypes (groups I and II) were determined by a newly developed C14 assay in which antibodies against group-specific recombinant proteins of the NS4 region were measured by ELISA (enzyme-linked immunosorbent assay). The genotypes determined by the C14 assay were compared to those determined by a polymerase chain reaction (PCR) in 50 patients. The HCV genotypes determined by both methods were consistent in 78% of the patients. The results of the assays were not contradictory in any patient. The sensitivity of the C14 assay was as high as over 90%. Frequency of HCV genotypes was studied in 300 patients with chronic liver diseases type C by using the C14 assay. The prevalence of groups I and that of II in patients with chronic hepatitis were 73% and 21%, the correspondence figures in those with liver cirrhosis 80% and 13%, and in those with hepatocellular carcinoma 81% and 13%, respectively. The clinical characteristics were similar between the patients with group I and II HCV infections in each disease category. In conclusion, the C14 assay is useful for epidemiological studies of HCV genotypes, and group I is a major HCV genotype of chronic liver diseases type C in Japan.