The coupling mechanism between depletion of Ca2+ stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to Ca2+ signaling in many cell types and has yet to be completely elucidated. Using Ca2+ release-activated Ca2+ (CRAC) channels in RBL-2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires Ca2+ and can be reversed by a diffusible Ca2+ influx factor. Thus the coupling between the depletion of Ca2+ stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low- molecular-weight, diffusible store-depletion signal.