The generation of GT-Ko mice has provided unique opportunities to study allograft and xenograft rejection in the context of anti-α1,3-Gal antibody (anti-Gal Ab) responses. In this study we used the allotrans-plantation model of C3H hearts into galactosyl-transferase-deficient (GT-Ko) mice and the xenotrans-plantation model of baby Lewis rat hearts into GT-Ko mice to investigate the ability of CTLA-4lg in combination with anti-CD40L mAb to control graft rejection and anti-Gal Ab production. Murine CTLA-4lg or anti-CD40L monotherapy prolonged allograft survival, and the combination of these reagents was most immuno-suppressive. However short-term treatment with murine cytotoxic T lymphocyte associated antigen-4 (muCTLA-4lg) and/or CD40 ligand (CD154) monoclonal antibodies (anti-CD40L mAbs) was unable to induce indefinite allograft survival. CTLA-4-immunoglobulin fusion protein (CTLA-4lg) or anti-CD40L monotherapy only marginally prolonged xenograft survival; the combination of human CTLA-4lg and anti-CD40L significantly prolonged xenograft survival (74days), while the combination of murine CTLA-4lg and anti-CD40L resulted in graft survival of >120days. CTLA-4lg or anti-CD40L monotherapy or the combination of these agents inhibited the production of alloAbs, including anti-Gal Abs. CTLA-4lg or anti-CD40L monotherapy partially controlled xenoAb and anti-Gal Ab production, while the combination was more effective. These observations corroborate our previous observations that humoral, including anti-Gal Ab, responses and rejection following allograft or concordant xenograft transplantation in GT-Ko mice are T-cell dependent and can be controlled by co-stimulation blockade.