Four monospecific antibodies against the hepatitis C virus nucleocapsid protein, which was expressed by recombinant baculovirus, were obtained by Epstein-Barr virus transformation of B cells from three patients with chronic hepatitis C virus infection. One of these antibodies was IgG and the other three were IgM. Their specificities were characterized initially by enzyme-linked immunosorbent assay and immunoblotting against hepatitis C virus proteins expressed by six recombinant baculoviruses with different hepatitis C virus sequence insertions. These specificities were confirmed, and their epitopes were more precisely determined with a series of overlapping decapeptides made by solid-phase pin technology. Two antibodies (1F4 and 2G6) reacted with the same peptides located near the amino(N)-terminus of nucleocapsid protein (amino acids 33-50). The third antibody (3B5) recognized the peptide consisting of amino acids 133-142, and the fourth antibody (3B9) was mapped to the carboxy(C)-terminus and reacted with a peptide consisting of amino acids 165-174. This epitope has not previously been reported. Two antibodies, 1F4 and 3B9, which are specific to the N-terminus and C-terminus of nucleocapsid protein, respectively, have been stably produced for more than 6 mo and are being subcloned to establish monoclonality. These antibodies should be useful reagents for the study of hepatitis C virus.