Xenoreactive antibodies are an integral part of the natural immune barrier to successful xenotransplantation between phylogenetically disparate species. Studies in primates suggest that the critical targets involved in hyperacute rejection of pig organs are glycoproteins expressed on endothelial cells and platelets. However, there is little information regarding the targets of xenoreactive antibodies and their cellular distribution in other experimental models of hyperacute xenograft rejection, including the commonly used guinea pig-to-rat model. The aim of this study was to characterize the target antigens on guinea pig platelets and endothelial cells that are recognized by rat anti-guinea pig antibodies. Using guinea pig platelet membrane proteins or intact guinea pig endothelial cells as antigens in an ELISA, we demonstrated that rat serum contains IgG and IgM anti-guinea pig antibodies; levels of antiplatelet antibodies correlated with those against endothelial cells. Serum from naive or complement-depleted rats transplanted with a guinea pig heart was investigated by immunoblotting against membrane proteins extracted from guinea pig platelets and endothelial cells. Normal rat serum revealed numerous bands on either platelet or endothelial cell immunoblots, without obvious similarities in banding pattern under reduced or nonreduced conditions. Absorption of rat serum with intact guinea pig endothelial cells reduced reactivity against numerous bands on endothelial cell membrane blots, but only partially reduced reactivity with three platelet bands (141, 155, and 210 kDa). Absorption of rat serum with intact guinea pig platelets resulted in reduction in reactivity against both platelet and endothelial cell immunoblots. Antibodies eluted from those intact guinea pig endothelial cells and platelets used to absorb rat sera were found to yield patterns of membrane blot reactivity similar to those with unabsorbed sera, suggesting that the proteins recognized are expressed on the cell surface. Lectin affinity blotting demonstrated many of the guinea pig endothelial cell and platelet membrane proteins to be glycosylated. However, digestion of endothelial cell and platelet immunoblots with a mixture of glycosidases failed to change the xenoreactivity with naive rat serum. Immunoblot analysis of serum samples taken daily from complement-depleted rats carrying a functioning guinea pig heart for 3-4 days showed an increase in intensity for specific protein bands on platelets (38, 48, 56,141, and 155 kDa) and endothelial cells (30, 210, and 270 kDa); no new protein bands were observed. Increased reactivity at several bands correlated with highly elevated rat serum anti-guinea pig antibody titers, as determined by ELISA. We conclude that (1) in contrast to the pig-to-primate species combination, anti-guinea pig antibodies in rats recognize membrane proteins of different size on endothelial cells and platelets; (2) although the proteins recognized on endothelial cells by anti-guinea pig antibodies have a different size pattern than those on platelets, there is evidence of shared antigenic determinants or crossreactivity; (3) carbohydrate substitutions on these xenoantigens do not appear to be major targets of rat anti-guinea pig antibodies; and (4) immune xenoreactive antibodies and natural antibodies in rat serum react with similar glycoproteins on guinea pig endothelial cell or platelet membranes.