STIM1 and Orail have recently been identified to be crucial in the regulation of store-operated Ca2+ entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orail in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We further demonstrate that Orail assembles into clusters that co-localize with STIM1 aggregations upon store depletion. The clustering of Orail is only seen when Oaril are co-expressed with STIM1, but not when expressed alone. Moreover, ER retreat from cell periphery leads to mismatching of Orail and STIM1 puncta. Therefore, we propose that store depletion causes aggregation and translocation of STIM1 in close apposition to the plasma membrane, which in turn recruits Orail in the plasma membrane to the sites of STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner. (c) 2006 Elsevier Inc. All rights reserved.