Critical to the prevention of xenograft loss is the prevention of delayed xenograft rejection (DXR), due to its resistance to conventional immunosuppression. The role of the carbohydrate galactose-alpha1,3-galactose (alpha1,3Gal) has been a matter of great debate and it has been proposed that the reaction between alpha1,3Gal epitopes on donor endothelial cells and recipient anti-alpha1,3Gal antibodies (Abs) may damage the graft during DXR. Recipient anti-alpha1,3Gal Abs are produced by CD4-dependent B cells. To test the above-mentioned hypothesis, hearts from alpha1,3Gal-free mice (GT-Ko mice), generated by alpha1,3-galacto-syltransferase gene disruption, were transplanted to anti-alpha1,3Gal antibody-free Lew/Mol rats. This model consists of an alpha1,3Gal/alpha1,3Gal-antibody-free environment, eliminating a possible influence of this specific system on DXR. A subgroup of recipients were furthermore CD4 depleted in order to inhibit CD4-dependent B-cell antibody production. Rejected hearts were evaluated by light- and immunofluorescence microscopy. Treatment effects on recipient T-cell subsets and cytokine expression were analyzed by flow cytometry, while antibody production was measured by ELISA. All recipients developed DXR with no differences among the groups. DXR was related to thrombosis with IgG and IgM desposition in vessel walls, as well as macrophage and granulocyte accumulation in the myocardium. No complement C3, CD4 cells or NK cells were found. Flow cytometric analysis confirmed peripheral blood CD4 depletion and IFN-gamma suppression in CD4 Ab-treated recipients. Finally, ELISA showed that specific anti-alpha1,3Gal Ab production was absent. However, Ab(s) against an unidentified Galalpha 1 were found among recipients. In our model, DXR is resistant to alpha1,3-galactosyltransferase gene inactivation and CD4 depletion. However, other Galalpha 1 epitopes and antibodies may play a role during DXR. Further studies are needed to elucidate the precise pathways leading to DXR.