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 trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD. 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 small molecular weight, diffusible store-depletion signal.