Natural killer (NK) cells have been thought to depend largely on perforin-mediated mechanisms for the induction of cell death in targets. However, this view has more recently been challenged. It is now clear that NK cells are capable of using death ligands like Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce cytotoxicity. Still, relatively little is known about the control of these "perforin-independent" cell death eliciting reactions, for example, the regulation of FasL expression on NK cells. In the present study, we confirm the ability of NK cells to mediate target cytotoxicity in the absence of perforin, in vivo and in vitro. We show that the induction of perforin-independent NK cell-mediated cell death is prevented by inhibiting signals mediated by MHC class I recognition. Furthermore, we demonstrate in vitro that cross-linking of the activation receptor NK1.1 on NK cells leads to the up-regulation of FasL on the cell surface. However, simultaneous engagement of an MHC class I binding inhibitory receptor prevents the externalization of FasL. These results provide a mechanistic explanation for theMHC class I-dependent regulation of perforin-independent cytotoxicity.