Combined immunotherapy with cyclophosphamide (Cy) and IL-12, but not IL-12 alone, stimulates eradication of a large established solid tumor (20 mm), MCA207, a methylcholanthrene-induced murine sarcoma. In these studies we demonstrate that NK1. 1+ cells and CD1d-dependent NK T cells each play important yet distinct roles in regression of a large tumor in response to Cy and IL-12, and we define a novel NK T cell subset, selectively increased by this treatment. Mice depleted of NK1. 1+ cells demonstrated more rapid initial tumor growth and prolonged tumor regression following treatment, but tumors were eventually eradicated. In contrast, initial tumor regression following therapy was unimpaired in CD1d−/− mice, which are deficient in most NK T cells, but tumors recurred. No tumor regression occurred following Cy and IL-12 therapy in CD1d−/− mice that were depleted of NK1. 1+ cells. We found that Cy and IL-12 induced the selective increase in liver and spleen lymphocytes of a unique NK T subpopulation (DX5+ NK1. 1− CD3+). These cells were not induced by treatment in CD1d−/− mice. Our studies demonstrate a contribution of both NK and NK T cells to the Cy-and IL-12-stimulated anti-tumor response. We describe the selective induction of a distinct NK T cell subset by Cy and IL-12 therapy, not seen following IL-12 therapy alone, which we suggest may contribute to the successful anti-tumor response induced by this immunotherapeutic regimen.