Natural killer (NK) cells play a crucial role in the antitumoral responses through cytolytic function and cytokine production. Expression of HLA-G at the surface of tumoral cells confers a protection against NK-cell cytolysis through its interaction with the ILT2 inhibitory receptor. Even though the role of this interaction on the inhibition of NK-cell cytotoxicity is well established, its effect on the molecular events occurring at the NK/target-cell synapse is not well characterized. We found that the interaction of the inhibitory receptor ILT2 with HLA-G inhibited the polarization of NK-cell lytic granules and the microtubule organizing center (MTOC) as well as the accumulation of filamentous actin (F-actin) at the area of contact. However, it did not affect the recruitment of the activatory receptor CD2 at the NK/target-cell interface. Even though CD2 was accumulated to the NK-cell synapse, the interaction of ILT2 with HLA-G efficiently inhibited intracellular calcium mobilization and IFN-gamma polarized production of NK cells. These results indicate that while the ILT2/HLA-G interaction leads to the inhibition of NK-cell functions, it displays differential effects on cytoskeleton reorganization and CD2 localization at the NK-cell synapse.-Favier, B., LeMaoult, J., Lesport, E., Carosella, E. D. ILT2/HLA-G interaction impairs NK-cell functions through the inhibition of the late but not the early events of the NK-cell-activating synapse.