Cytochromes c have been used as antigens in a murine T-lymphocyte proliferation assay in order to characterize the nature of determinants whose recognition is under immune response (Ir) gene control. The cytochromes are advantagous as antigens because 1) they have well-characterized primary and tertiary structures, 2) they are antigenically simple, differing from mouse cytochrome c at only a small number of amino acid residues, and 3) there exist a large number of evolutionary variants which can be used to locate antigenic sites by cross-stimulation. In the present studies, the T-lymphocyte proliferative response to pigeon cytochrome c was shown to be under the control of two complementing major histocompatibility (MHC)-linked Ir genes in mice of the H-2a and H-2k haplotypes. Mice of the H-2b, H-2d, H-2p, H-2q, H-2s, and H-2u haplotypes were low or nonresponders. Complementation was demonstrated by showing that an F1 hybrid between two nonresponder recombinant strains, B10.A(4R) and B10.A(5R), could respond to pigeon cytochrome c. The determinant on the cytochrome recognized in this immune response was located to the C-terminal portion of the molecule around residues 89 and/or 100. This was shown by the failure of closely related cytochromes from the Pekin duck and chicken to cross-stimulate T lymphocytes immune to pigeon cytochrome; position 89 and 100 carry the only residues different from those in mouse cytochrome c that are unique to pigeon cytochrome among the three bird cytochromes tested. This localization was further substantiated by demonstrating that the cyanogen bromide cleavage-fragment (residues 81-104) from pigeon cytochrome, but not the same fragment from Pekin duck cytochrome, was as good a stimulant of T cells immune to the whole molecule as the intact cytochrome. These results identify the immunogenic site on the molecule as one which differs from mouse cytochrome c by only one or two amino-acid residues. Thus, T-cell immune responses, which are under MHC-linked Ir gene control, are as capable as antibody responses of recognizing subtle differences in protein structure. However, the ability of T cells to respond equally well to stimulation with polypeptide fragments or with the whole molecule suggests either that T-cell recognition involves certain differences from B cell recognition or that in some cases the fragments possess a similar spatial structure to that of the corresponding segment in the native protein.