STIM1 and CRACM1 (or Orai1) are essential molecular components mediating store-operated Ca²⁺ entry (SOCE) and Ca²⁺ release-activated Ca²⁺ (CRAC) currents. Although STIM1 acts as a luminal Ca²⁺ sensor in the endoplasmic reticulum (ER), the function of STIM2 remains unclear. Here we reveal that STIM2 has two distinct modes of activating CRAC channels: a store-operated mode that is activated through depletion of ER Ca²⁺ stores by inositol 1,4,5-trisphosphate (InsP₃) and store-independent activation that is mediated by cell dialysis during whole-cell perfusion. Both modes are regulated by calmodulin (CaM). The store-operated mode is transient in intact cells, possibly reflecting recruitment of CaM, whereas loss of CaM in perfused cells accounts for the persistence of the store-independent mode. The inhibition by CaM can be reversed by 2-aminoethoxydiphenyl borate (2-APB), resulting in rapid, store-independent activation of CRAC channels. The aminoglycoside antibiotic G418 is a highly specific and potent inhibitor of STIM2-dependent CRAC channel activation. The results reveal a novel bimodal control of CRAC channels by STIM2, the store dependence and CaM regulation, which indicates that the STIM2/CRACM1 complex may be under the control of both luminal and cytoplasmic Ca²⁺ levels.—Parvez, S., Beck, A., Peinelt, C., Soboloff, J., Lis, A., Monteilh-Zoller, M., Gill, D. L., Fleig, A., Penner, R. STIM2 protein mediates distinct store-dependent and store-independent modes of CRAC channel activation.