As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx plays an important role in Ca2+ signaling and many other cellular processes1–3. A calcium release-activated calcium (CRAC) channel in T lymphocytes is the best characterized SOC influx channel4–6 and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation7–10. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously, we identified Stim and the mammalian homolog STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes11. Here, we show that expression of EF hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization, and surface biotinylation we demonstrate that STIM1 migrates from ER-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.