There are two distinct pathways by which peptide antigens are recognized in the process of allograft rejection. The "direct" pathway involves presumably myriad host peptides recognized in intact allo MHC molecules expressed by donor antigen presenting cells, while the "indirect" pathway involves donor derived peptides processed and presented by host APC. We hypothesize that the direct pathway is characterized by a high precursor frequency, but generally have a low TCR avidity. In contrast, the indirect pathway initially has low precursor frequency, but potentially high TCR avidity. Since the conventional mixed lymphocyte reaction (MLR) probably represents primarily the direct pathway, we examined the response characteristics of MLR and OVA peptide specific transgenic TCR cells that have both conventional self MHC/peptide reactivity and direct alloreaction to H-2b APC. The sensitivity to adhesion molecule inhibition (anti-LFA-1 and anti-CD4) and sensitivity to cyclosporin A were examined in direct alloresponses and with various peptide doses for conventional self MHC/peptide reactivity. The data demonstrate that conventional MLR and the direct allorecognition of the transgenic TCR cells and low peptide dose in self MHC all are much more sensitive to inhibition with all three agents than the same transgenic TCR cells with high peptide dose. These data are consistent with our hypothesis and suggest that the emergence of a high avidity, indirect alloantigen specific T cell clone is a key event in the development of chronic transplant rejection.