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 microtu‐bule 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‐γ 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. FASEB J. 24, 689–699 (2010). www.fasebj.org