Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) influx in platelets and other cells activated by a reduction in Ca(2+) concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca(2+), although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca(2+). Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn(2+) entry, which was inhibited by extracellular Ca(2+) in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25-139 of PM-STIM1 that contains the Ca(2+)-binding domain, prevented the inactivation of Ca(2+) entry induced by extracellular Ca(2+). TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca(2+)-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca(2+), an event that is likely to be mediated by interaction with Orai1.