Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.