NK‐cell function is regulated by a balance between inhibitory and activating killer cell immunoglobulin‐like receptors (KIR) that specifically recognize HLA class I molecules. Using KIR‐specific mAb to discriminate between KIR2DS1 and KIR2DL1 receptors, we show that KIR2DS1⁺ NK cells are C2‐alloreactive only from C2⁻ individuals. Moreover, using an in vitro model of NK‐cell expansion, we show here that the frequency of KIR2DL1⁺ NK cells is significantly higher in the absence of C2 ligand on stimulator EBV‐B cells than in its presence. This observation was made regardless of the presence or absence of the autologous C2 ligand, suggesting that the C2⁻ EBV‐B stimulator cells used in this in vitro model could activate unlicensed KIR2DL1⁺ NK cells. In the case of KIR2DL1⁺/S1⁺ genotyped individuals, KIR2DS1⁺ NK‐cell frequency was increased after stimulation with C2⁺ compared with C2⁻ stimulator B cells, but only from C2⁻ individuals. Altogether, these data highlight the C2 alloreactivity of KIR2DS1⁺ NK cells that is only observed in C2⁻ individuals. These results provide new insights into the way in which NK KIR cell expansion might be regulated in an allogeneic environment.