As the sole Ca(2+) entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca(2+) signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca(2+) store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca(2+) store. We propose that STIM1 functions as the missing link between Ca(2+) store depletion and SOC influx, serving as a Ca(2+) sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.