Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.