Of the antigenic determinants so far identified for cytochrome c, only one involves more than a single amino acid substitution between the immunogen and host proteins. Both a threonine at position 89 and a glutamic acid at position 92 control one of the three antigenic sites identified in horse cytochrome c, as expressed in rabbits. Three antibody subpopulations, all directed against this region of the molecule, were isolated from the serum of a single rabbit by adsorption on a series of insolubilized cytochromes c. Antibody fluorescence quenching titrations with a variety of cytochromes c were used to confirm the identification of the antigenic determinant and to examine the subtle differences in the specificities of the three subpopulations. The determinant in the region of Residues 89-92 is affected by amino acid substitutions at positions 88 and 96. Since all these residues are in an alpha-helix the farthest distance between them is only 12 A and therefore, the Residues 88-96 can all be accommodated in the antibody binding site. The ability to identify and describe the antigenic determinant, as well as separate subpopulations directed against this site, demonstrates the resolution possible using a series of homologous protein antigens.