Store-operated Ca 2+ entry is mediated by Ca 2+ release-activated Ca 2+ (CRAC) channels following Ca 2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca 2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.Receptor-mediated signaling in nonexcitable cells, immune cells in particular, involves an initial rise in intracellular Ca 2+ due to release from the intracellular stores. The resulting depletion of the intracellular stores induces Ca 2+ entry through the plasma membrane through CRAC channels (1-4). This phenomenon is central to many physiological processes such as T cell proliferation, gene transcription, and cytokine release (3, 5-7). Biophysically, CRAC currents have been well characterized (2,8,9), but the identity of the CRAC channel itself and the pathway resulting in its activation are still unknown. Recently, STIM1 (for stromal interaction molecule in Drosophila) was identified as an essential component of store-operated calcium entry (10,11). This protein is located in intracellular compartments that likely represent parts of the endoplasmic reticulum (ER). It has a single transmembranespanning domain with a C-terminal Ca 2+ -binding motif that appears to be crucial for its hypothesized function as the ER sensor for luminal Ca 2+ concentration. When stores become depleted, STIM1 redistributes into distinct structures (punctae) that move toward Fig. 1, B and C, from cells treated with dsRNA against Rho1 (mock) and stim1, as well as two genes we later identified as CRAC modulators 1 and 2 (CRACM1 and CRACM2). On the basis of inhibitory efficacy relative to positive and negative controls, we identified ~1500 genes that reduced Ca 2+ influx to varying degrees (table S1). After eliminating numerous genes based on artifactual fluorescence signals or because they represent known housekeeping genes, cell cycle regulators, and so on, we eventually arrived at 27 candidate genes (table S2) that were subsequently evaluated in a secondary screen using single-cell patch-clamp assays.From the secondary patch-clamp screen, we identified two novel genes that are essential for CRAC channel function, CRACM1 (encoded by olf186-F in Drosophila and FLJ14466 in human) and CRACM2 (encoded by dpr3 in Drosophila, with no human ortholog). We measured CRAC currents in Drosophila Kc cells after inositol 1,4,5-trisphosphate (IP 3 )-mediated depletion of Ca 2+ from intracellular stores. Both untreated control wild-type cells and cells treated with an irrelevant dsRNA against Rho1 (mock) responded by ra...