BACKGROUND: Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically-modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti-pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8(+) and gammadelta T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co-stimulation of T cells. METHODS: Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell-cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D-Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. RESULTS AND CONCLUSIONS: In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.