Natural Killer (NK) cell activation requires the integration of inhibitory and activating signaling. Inhibitory signals are determined by members of the killer cell immunoglobulin-like receptor (KIR) family, which have major histocompatibility complex (MHC) class I ligands. Loss of this inhibitory signal leads to NK cell activation. Thus, down-regulation of MHC I during viral infection or cancer induces NK cells activation. However, NK cell activation in the presence of MHC-I has been demonstrated for HLA-C*0102 through changes in its peptide content: “peptide antagonism”. Here we identify an antagonist peptide for HLA-C*0304 suggesting that peptide antagonism is a generalizable phenomenon and, using a combination of mathematical modelling, confocal imaging, and immune-assays, we quantitatively determine mechanisms that underlie peptide antagonism in inhibitory KIR2DL2/3 signaling. These data provide a mechanism for NK cell activation based on a reduction of inhibitory signaling in the presence of preserved levels of MHC class I.