Engagement of major histocompatibility complex (MHC) class I molecules on immune cells, where they are usually highly expressed, induces signal transduction events of unclear significance. We show here that antibody-mediated cross-linking of MHC-I molecules on human natural killer (NK) cells inhibits their cytotoxic activity against tumor target cells. Inhibition by anti-MHC class I monoclonal antibody exhibits molecular specificity and is an isotype and Fc-independent process. Physical hindrance of specific molecular recognition, induction of apoptosis, or reciprocal NK cell killing, which could be induced by cross-linking of MHC I molecules, has also been ruled out as putative mechanisms of inhibition. Confocal microscopy analysis revealed that MHC class I molecules on the surface of NK cells colocalize constitutively with GM1, a marker of lipid rafts. Cross-linking of MHC class I resulted in the asymmetric redistribution of GM1-enriched raft domains, which are concentrated to the immunological synapse, and MHC I molecules, which segregate to the opposite pole. Also, the cross-linking of MHC I on NK cells induced intracellular tyrosine phosphorylations. These results suggest that MHC I molecules on NK cells could transmit inhibitory signals upon engagement with putative ligands expressed on the surface of those cells that need to be protected from natural cytotoxicity.