Heterotopic cardiac xenotransplantation from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine to baboons was performed to characterize immunological reaction to the xenograft in the absence of anti-Gal antibody-mediated rejection. Eight baboons received heterotopic cardiac xenografts from GalT-KO porcine donors. All baboons were treated with chronic immunosuppressive therapy. Both histological and immunohistochemical studies were performed on biopsy and graftectomy samples. No hyperacute rejection was observed. Three baboons were euthanized or died 16 to 56 days after transplantation. The other five grafts ceased beating between days 59 and 179 (median, 78 days). All failing grafts exhibited thrombotic microangiopathy (TM) with platelet-rich fibrin thrombi in the microvasculature, myocardial ischemia and necrosis, and focal interstitial hemorrhage. TM developed in parallel with increases in immunoglobulin (IgM and IgG) and complement (C3, C4d, and C5b-9) deposition, as well as with subsequent increases in both TUNEL(+) endothelial cell death and procoagulant activation (increased expression of both tissue factor and von Willebrand factor and decreased expression of CD39). CD3(+) T-cell infiltration occurred in all grafts and weakly correlated with the development of TM. In conclusion, although the use of GalT-KO swine donors prevented hyperacute rejection and prolonged graft survival, slowly progressive humoral rejection--probably associated with non-Gal antibodies to the xenograft--and disordered thromboregulation represent major immunological barriers to long-term xenograft survival.