There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2(+) NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2(+) NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2(+) NK cells and accelerated islet graft failure. The Ly49i2(+) NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1(+) NK cells infiltrated the grafts, but nonalloreactive NKR-P1B(+) cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.