Previous studies has shown that human anti-pig reactivity in mixed lymphocyte cultures require the indirect presentation of antigens by human antigen presenting cells (APC). Xenoreactivity was inhibited by blockade of human costimulatory molecules. We investigated the role of porcine costimulatory molecules in their ability to activate human T cells directly. Porcine CD80 was cloned from lipopolysaccharide (LPS)-activated porcine lymphocytes. Sequence analysis showed a high degree of conservation in residues involved in CD28/CTLA4. COS cells transfected with porcine CD80 was able to activate human T cells in a cyclosporine independent manner, demonstrating that porcine CD80 can costimulate human T cells. Tumor necrosis factor-α (TNF-α) activated porcine splenocytes have been shown to up-regulate B7s. In order to test the effect of costimulation blockade in a xeno system, activated splenocytes were cultured with purified CD4+ T cells. The results demonstrated that these cells were capable of activating human T cells and this activation can be blocked by using an antihuman CD80 antibody that demonstrated cross-reactivity to porcine CD80. Non-cross reactive antibodies had no effect, again suggesting direct activation of the human T cells. These data suggest that a reagent that can block both the direct and indirect activation is necessary for a discordant xenotransplant.