Recent animal studies suggest that indirect T-cell recognition of alloantigen plays an important role in allograft rejection and tolerance. In this study, we generated T cell clones from Lewis (LEW, RT1(l)) rat lymph node cells that had been primed in vivo by immunization with immunogenic class II MHC allopeptide RT1.D(u)beta2, representing residues 20-44 of the polymorphic beta chain of RT1.Dbeta(u) (Wistar Furth [WF]). Using reverse transcriptase polymerase chain reaction transcript analysis with specific rat T cell receptor Vbeta primers, we show that six out of nine T cell clones specifically proliferated to RT1.D(u)beta2 and expressed Vbeta 9. One of these clones, clone 2F4, which specifically proliferated to RT1.D(u)beta2 in a dose-response fashion and produced interferon-gamma in response to restimulation by RT1.D(u)beta2, was selected for further studies. The beta-chains of RT1.D(l) and RT1.D(u) residues 20-44 differ by two amino acids at positions 30 and 38. We synthesized two peptides, each containing a single polymorphic site: RT1.D(u)beta 20-33 and RT1.D(u)beta 31-44. Both these peptides were immunogenic by LEW responders, since lymph node cells primed by immunization proliferated equally to the peptides in vitro. Interestingly, in vitro dose-response studies with clone 2F4 showed better proliferative response to peptide RT1.D(u)beta 20-33 than to peptide RT1.D(u)beta 31-44, indicating that this T cell clone preferentially recognizes a single amino acid difference on residue 30. Finally, it has been suggested that indirect allorecognition by CD4+ T cells mediate graft rejection by delayed-type hypersensitivity responses, although definitive studies are lacking. Systemic injection of the 2F4 clone to naive LEW rats elicited an antigen-specific delayed-type hypersensitivity response against RT1.D(u)beta2 peptide and WF splenocytes, confirming indirect presentation in vivo. These data demonstrate that Th1 cell clones generated by in vivo priming via the indirect pathway utilize specific T cell receptor Vbeta and recognize single amino acid differences in the allopeptide. More importantly,these Th1 clones are capable of mediating a specific immune response in vivo. These studies with MHC allopeptide-specific T cell clones further delineate the cellular mechanisms of indirect allorecognition and provide a potential strategy to study its role in acute and chronic rejection, and tolerance.