To locate epitopes on internal antigens of rabies virus, purified N and NS proteins of the nucleocapsid were cleaved at methionine, tryptophan or glutamic acid residues, transferred to nitrocellulose and immunostained using monoclonal antibodies (MAbs) specific for N and NS proteins, respectively. Five MAb-positive fragments of N protein and one fragment of NS protein were located after NH2-terminal amino acid sequence analysis within the deduced amino acid sequences of N and NS proteins. Antigenic analysis of synthetic overlapping peptides corresponding to the amino acid sequences of these fragments localized two major antigenic sites of N protein and one antigenic site of NS protein. Like the N- and NS-specific MAbs, anti-peptide antisera produced against the different synthetic antigens either reacted in a type-common fashion with all rabies virus strains, or in a type-specific manner with a restricted number of strains. The synthetic peptides corresponding to the three antigenic regions of the N and NS proteins also stimulated proliferation of human T lymphocytes derived from vaccinees who received inactivated rabies virus vaccine. This suggested that the antigenic regions of N and NS proteins are recognized by both B and T cells.