In the pig-to-primate model of xenotransplantation, graft rejection is initiated by binding of the recipient's xenoreactive natural antibodies (XNA), mainly of the IgM type, to antigens constitutively expressed on donor endothelial cells (EC). As a consequence of XNA binding and complement fixation, the EC become activated, which is considered to be a major mechanism promoting hyperacute as well as later phases of graft rejection. It is not clear whether binding of XNA to activated EC also contributes to delayed rejection. We asked whether EC activation by cytokines results in the expression of other novel surface antigens recognized by XNA which might become relevant in progressive stages of graft rejection. We activated porcine aortic EC and smooth muscle cells with tumor necrosis factor (TNF), interleukin 1, or lipopolysaccharide and studied expression of new XNA-binding antigens. Expression of two glycoproteins, gp65 and gp100, was strongly induced by recombinant human TNF in EC but not in smooth muscle cells. Notably, gp100 expression was specific to TNF activation, whereas gp65 could also be induced by interleukin 1 or lipopolysaccharide. Cell surface labeling indicated that gp65 is expressed on the plasma membrane. Recognition of XNA-binding antigens on resting EC occurs via alpha-galactosyl epitopes. In contrast, gp65 and gp100 were recognized independently of this epitope. Our data show that gp65 and gp100 represent selective cytokine-induced markers on EC that may have importance in a porcine-to-primate model of xenotransplantation. Conceivable functions of gp65 and gp100 are discussed.