Antibodies directed against galactose-alpha1,3-galactose (alphaGal) are believed to play an important role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether alpha1,3-galactosyltransferase-deficient (Gal KO) mice can naturally acquire a sufficient anti-alphaGal titre to cause the delayed type rejection of alphaGal-expressing hearts. Gal KO mice of various ages were assessed for anti-alphaGal antibody levels. alphaGal-expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. In Gal KO mice greater than 6-month-old, 64% had an anti-alphaGal antibody titre above the background level. When wild-type alphaGal-expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti-alphaGal antibodies with age. The titre in these mice was sufficient to cause a "delayed-type" rejection of a significant proportion of alphaGal-expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti-alphaGal antibodies in the pathogenesis of DXR.