The region of the horse cytochrome c molecule recognized by Mab SJL2-4 specific for the denatured form of the protein was located around residues 22-28. Binding studies on antigen pulsed macrophages were also performed. Surprisingly, heme peptide 1-65 was not recognised by Mab when bound on macrophages. This correlates with the incapacity of the same peptide to activate the T-cell clone 2-16. Binding sites on antigen pulsed macrophages varied between 0.5-2 x 10(6) per cell depending on the conditions used. The expression of the antigenic determinant as detected by Mab was also followed under different conditions (chloroquine, trypsin treatment) and time. Kinetics parameters of the antigen-antibody reaction in solution and on antigen bound macrophages were also determined and are dramatically different. This is correlated with a different structure of the peptide in solution and on macrophage cell surface.