Recent reports have demonstrated that humoral factors, especially antibodies elicited by xenoantigens, play an important role in the rejection of concordant cardiac xenografts. These induced antibodies, however, have not been well characterized. Therefore, we investigated the rejection mechanism, especially the role of humoral immunological responses in the concordant rat to mouse cardiac xenograft model. Lewis rat hearts transplanted into C3H/HeN mice were rejected in 5-6 days. The essential role of humoral factors in the rejection was demonstrated by histological analysis of the rejected hearts showing interstitial hemorrhage, scant cellular infiltration, and the dense deposition of mouse IgG, IgM, and C3 on the graft endothelial cells. In addition, mice that received hyperimmune serum (serum at the 10th day after transplantation) rejected rat hearts hyperacutely. Flow cytometrical analysis using cultured donor rat coronary endothelial cells demonstrated the xenoreactive antibodies of all subclasses, but especially strong reactivity of IgM and IgG2a in the serum at rejection. These xenoreactive antibodies were produced against not only MHC, but also non-MHC antigens on graft endothelial cells. In vivo depletion of L3T4+ T cells led to the suppression of xenoreactive antibody production and the prolongation of graft survival, indicating that antibody production in this model needs L3T4+ T cell help.