Store-operated Ca2+ entry (SOCE) is a universal mechanism to increase intracellular Ca2+ concentrations in non-excitable cells. It is initiated by the depletion of ER Cal' stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca2+ release activated Cal' (CRAG) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca2+ influx and CRAC currents (I-CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate ICRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca2+ stores. (C) 2009 Elsevier Inc. All rights reserved.