The putative core gene of hepatitis C virus (HCV) was incorporated into a plasmid vector (pCC5-J4), and expressed in Escherichia coli. The product of 180 amino acids (p20c) was purified by gel electrophoresis in the presence of sodium dodecyl sulfate, and used in enzyme-linked immunosorbent assay for antibodies against the putative core protein of HCV (anti-p20c). Anti-p20c was detected in 13 (1.5%) of 873 apparently healthy blood donors. It was detected in 205 (86.5%) of 237 patients with acute or chronic non-A, non-B (NANB) hepatic disease, significantly more frequently (p less than 0.01) than antibodies against the C100-3 protein encoded by nonstructural regions of HCV (anti-C100-3) that was found in 178 (75.1%). Anti-p20c developed in the circulation of a patient with acute NANB hepatitis much earlier than anti-C100-3. HCV RNA was detected by polymerase chain reaction in serum samples from blood donors positive for anti-p20c in high titers, one of which was negative for anti-C100-3. These results indicated that anti-p20c would be useful in complementing anti-C100-3 for the diagnosis of NANB hepatitis and further decreasing the incidence of posttransfusion NANB hepatitis.