Natural killer and T cells express at their surface, members of a multigenic family of killer cell inhibitory receptors (KIR) for major histocompatibility complex Class I molecules. KIR engagement leads to the inhibition of natural killer and T cell activation programs. We investigated here the functional reconstitution of KIR in a non-lymphoid cell type. Using stable transfection in the RBL-2H3 mast cell line, we demonstrated that (i) KIR can inhibit signals induced by FcepsilonRIgamma or CD3zeta polypeptides that bear immunoreceptor tyrosine-based activation motifs; (ii) two distinct immunoreceptor tyrosine-based inhibition motifs-bearing receptors, i.e. KIR and FcgammaRIIB, use distinct inhibitory pathways since KIR engagement inhibits the intracellular Ca2+ release from endoplasmic reticulum stores, in contrast to FcgammaRIIB, which only inhibits extracellular Ca2+ entry; (iii) KIR require co-ligation with an immunoreceptor tyrosine-based activation motif-dependent receptor to mediate their inhibitory function. This latter finding is central to the mechanism by which KIR selectively inhibit only the activatory receptors in close vicinity. Taken together our observations also contribute to define and extend the family of immunoreceptor tyrosine-based inhibition motif-bearing receptors involved in the negative control of cell activation.