The T-cell response to xenografts is induced by direct and indirect recognition of xenoantigens. Although the importance of indirect recognition is well established in vitro, the contribution of this pathway to xenograft rejection in vivo remains to be fully elucidated. We herein investigated the direct contribution of indirect recognition to cardiac xenograft rejection in the rat-to-mouse (PVG.R8-to-C57BL/10) concordant model. Rat xenoantigens invoked a vigorous proliferative response in mouse T cells harvested from naïve or graft recipients at rejection. Indirect recognition predominated the response, as antibodies against mouse class II I-A(b), CD80, or CD86 molecules significantly (45 to 60%) blocked the proliferative response. Importantly, the blockade of indirect recognition in vivo by treating the graft recipients with a monoclonal antibody (mAb) against class II I-A(b) molecule on days 0, 1, and 3 post-transplantation resulted in significant (P < 0.009) prolongation of cardiac xenograft survival (Mean Survival Time (MST) >94 +/- 55 days vs. 7 +/- 0.8 days for controls). In contrast, treatment of recipients with a mAb against mouse class I H-2K(b)/D(b) molecules did not significantly affect graft rejection (MST = 8 +/- 1 days). These results demonstrate that indirect recognition mediated by CD4(+) T cells plays a critical role in the rejection of cardiac grafts in the rat-to-mouse xenogeneic model.