We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col (V)), and that col (V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col (V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col (V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col (V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1 lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1 l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col (V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+ CD25− and produced IFN-γ in response to col (V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col (V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo-and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col (V).