Killer cell Ig-like receptors (KIRs) regulate the function of human natural killer and T cell subsets. A feature of the KIR locus is the clustering of homologous genes encoding for inhibitory and activating KIR. Inhibitory and activating KIR differ for ligand specificities and/or affinities. In particular, we show here with KIR tetramers that activating KIR2DS2 does not bind HLA-Cw3 molecules recognized by inhibitory KIR2DL2, despite 99% extracellular amino acid identity. We also report the 2.3-Å structure of KIR2DS2, which reveals subtle displacements of two residues (Tyr45 and Gln71) involved in the interaction of KIR2DL2 with HLA-Cw3. These results show that KIR molecules cannot tolerate any variability in their three-dimensional structure without altering their MHC class I recognition capacities. Therefore, the mode of recognition used by KIR largely differs from the conformational changes that characterize T cell receptor or NKG2D interaction with their respective ligands.