A bank of mouse monoclonal antibodies has been produced with reactivity to gonococcal pili to investigate epitopes of the pilus structural protein, pilin. Pili of Neisseria gonorrhoeae strains R10 and MS11 were used as immunogens to elicit 19 monoclonal antibodies reactive with the homologous pili type in ELISA. Of the 19 antibodies, 16 demonstrated type-specific reactivity and 3 were cross-reactive with heterologous pili. Reactivity of the antibodies with the carboxyterminal, cyanogen bromide fragment (CB-3) of R10 pilin allowed their classification into three groups. The first group (10 antibodies) were R10 specific and equally reactive with the R10 CB-3 fragment. The second group (6) were also type specific but demonstrated poor reactivity with the CB-3 fragment. This suggested that the epitopes of the first group are linear, and those of the second group, nonlinear. The third group (3), consisting of the cross-reactive antibodies, were not reactive with the CB-3 fragment. Two of the antibodies in group 3 were examined in detail to localize their epitopes. The epitope of one, 9B9/H5, was shown to be a linear determinant. This antibody was reactive with a fragment of MS11 pilin (residues 31-111) and to a synthetic peptide representing residues 69-84 in MS11 pilin. The epitope was more finely mapped, with shorter synthetic peptides conjugated to bovine serum albumin, to an eight amino acid segment (residues 69-76). The epitope of 1E8/G8, a strongly reactive antibody, proved elusive to this type of analysis and probably results from conformational restraints. The significance of species-specific epitopes in the pilin protein is discussed.