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 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 are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by ciclosporine A immunosuppression. Also alloreactive KLRH1⁺ NK-cells infiltrated the grafts, but non?alloreactive 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.