The antigen fine specificity of T cell hybridomas recognizing the horse apocytochrome c fragment 1-65, restricted to the I-Ab molecule, was determined to gain some insight into the molecular nature of T cell antigenic peptides. Two major groups of clones specific for distinct subsites, namely residues 1-38 and 39-65, could be identified. Hybridomas recognizing the latter determinant were further tested with different horse cytochrome c peptides and analogues. This analysis revealed the presence of at least two epitopes encompassed by residues 47-53 and 48-53. Furthermore, clones specific for the amino acid sequence 48-53 showed considerable heterogeneity in respect to the antigen concentration required to obtain 50% of the maximal interleukin 2 secretion. Most prominent was the heteroclitic response towards tuna cytochrome c which differs at positions 44, 46 and 47 from the horse cytochrome c molecule in the relevant region. Comparison of the conformation of the sequence 43-46 between the two cytochrome c suggests that this segment, which forms a 3(10) bend, may be important in maintaining the proper structure of the antigenic determinant. Moreover, the variations up to 180-fold in the concentrations of the cross-reacting cytochrome c and peptides required for stimulation were not always correlated with the maximal interleukin 2 secretion they induced. This indicates that the biological response, that is supposed to be an indication of the affinity of the T cell receptor for its ligand, is not necessarily a function of the antigen concentration.