There are two distinct pathways by which peptide antigens that 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. Since the conventional mixed lymphocyte reaction probably represents primarily the direct pathway and often fails to correspond to specific patterns of unresponsiveness in allograft tolerance mice, we reasoned that the relevant antigens after the initial perioperative period for rejection vs tolerance were indirect alloantigens. To find peptides which are recognized in the indirect pathway by haplotype H-2b cells, 15-mer of overlapping peptides corresponding to variable regions of class I and class II MHC molecules of H-2d and H-2k haplotypes were synthesized in a 3-amino acid-step manner. In addition, a constant peptide of Eα (56-73) shared by Ek and Ed allelles that is known to bind to I-Ab was synthesized. These peptides were screened by measuring the proliferative response of nylon wool purified spleen and lymph node T cells from C57BL/6 mice after in vivo immunization with B10.D2 (H-2d) or B10.BR (H-2k) spleen cells. Cultures were set up in 0.5% mouse serum containing medium stimulated with peptide plus irradiated C57BL/6 spleen cells. Several peptides stimulated specific proliferative responses, including H-2Kd (87-101) and Eα (56-73). Although these peptides can stimulate in vivo immunized T cells, immunization with Eα (56-73) in CFA did not accelerate either skin or vascularized cardiac allografts.