Modulation of human NK cell function by killer cell Ig-like receptors (KIR) and MHC class I is dominated by the bipartite interactions of inhibitory lineage III KIR with the C1 and C2 epitopes of HLA-C. In comparison, the ligand specificities and functional contributions of the activating lineage III KIR remain poorly understood. Using a robust, sensitive assay of KIR binding and a representative panel of 95 HLA class I targets, we show that KIR2DS1 binds C2 with~ 50% the avidity of KIR2DL1, whereas KIR2DS2, KIR2DS3, and KIR2DS5 have no detectable avidity for C1, C2, or any other HLA class I epitope. In contrast, the chimpanzee has activating C1-and C2-specific lineage III KIR with strong avidity, comparable to those of their paired inhibitory receptors. One variant of chimpanzee Pt–KIR3DS2, the activating C2-specific receptor, has the same avidity for C2 as does inhibitory Pt–KIR3DL4, and a second variant has~ 73% the avidity. Chimpanzee Pt–KIR3DS6, the activating C1-specific receptor, has avidity for C1 that is~ 70% that of inhibitory Pt–KIR2DL6. In both humans and chimpanzees we observe an evolutionary trend toward reducing the avidity of the activating C1-and C2-specific receptors through selective acquisition of attenuating substitutions. However, the extent of attenuation has been extreme in humans, as exemplified by KIR2DS2, an activating C1-specific receptor that has lost all detectable avidity for HLA class I. Supporting such elimination of activating C1-specific receptors as a uniquely human phenomenon is the presence of a high-avidity activating C1-specific receptor (Gg–KIR2DSa) in gorilla.