An enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of hepatitis C virus (HCV) infection, using HCV core protein (p22) synthesized by a recombinant baculovirus. Among 58 clinically well-defined chronic non-A, non-B hepatitis (NANBH) patients, 49 (84.5%) were positive for p22 antibody (anti-p22), whereas 42 (72.4%) were positive for C100-3 antibody (anti-C100-3), as measured by the present assay using the HCV nonstructural protein as antigen. Thirty-nine patients (67.2%) had both antibodies. No significant level of anti-p22 was detected in sera of chronic hepatitis B patients or normal blood donors. In typical post-transfusion NANBH patients, anti-p22 could be detected at, or even before, the first alanine aminotransferase peak. Anti-p22 was also detected in blood donors who were previously shown to be involved in transmitting HCV but in whose serum anti-C100-3 was not detectable. The ELISA detecting antibody to the HCV core protein expressed and properly processed in animal cells will be useful for mass screening of donor blood as well as for early diagnosis of hepatitis C.