Abstract NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2–expanded NK cells was further up- or downregulated by IL-12 or TGF-β, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti–CTLA-4 mAbs.