Store-operated Ca²⁺ entry (SOCE), a process activated by depletion of intracellular Ca²⁺ stores, is a major pathway for Ca²⁺ influx in non-excitable cells. Ca²⁺ store depletion in platelets results in de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP₃RII) to the Ca²⁺ entry channel hTRPC1 (Rosado & Sage, 2000), an event supported by remodelling of the actin cytoskeleton (Rosado & Sage, 2001). Recently, stromal interaction molecules (STIMs) have been identified as intraluminal Ca²⁺ sensor proteins involved in the regulation of SOCE (Liou et al. 2005; Zhang et al. 2005). Here we have investigated the interaction of STIM1 with hTRPC1 and its regulation by the actin cytoskeleton upon depletion of the Ca²⁺ stores in human platelets. Blood was drawn from healthy, drug free volunteers. Cytoskeletal association of STIM1 in platelets was determined by Western blotting and interaction between STIM1 and hTRPC1 was investigated by co-immunoprecipitation as described previously (Rosado & Sage, 2000). Determination of cell surface expression of STIM1 was performed by immunocytochemistry. Co-immunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with STIM1 in resting platelets suspended in a medium containing 200 µM Ca²⁺ or in a Ca²⁺-free medium. Depletion of the intracellular Ca²⁺ stores, by treatment for 3 min with thapsigargin (TG; 1 µM) in combination with ionomycin (Iono; 50 nM), significantly enhanced the association of STIM1 with hTRPC1 to 153% of control, both in the absence and presence of extracellular Ca²⁺ (p<0.05; Student’s t test; n=8). Similar results were obtained in BAPTA-loaded cells, suggesting that an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) is not required for the association of STIM1 with hTRPC1. Treatment with TG + Iono increased STIM1 cell surface localization to 250% of control (p<0.05; n=6). A small amount of STIM1 (6%) was found to be associated with the cytoskeletal fraction in resting platelets. Cytoskeletal association was significantly increased after treatment with TG + Iono to 195% of control (p<0.05). Platelet treatment for 40 min with 10 µM cytochalasin D, to prevent actin filament polymerization (Rosado & Sage, 2001), significantly reduced the association of STIM1 with hTRPC1 in resting platelets by 80 % and abolished the enhanced interaction between STIM1 and hTRPC1 induced by TG + Iono (p<0.05; n=6). Our results indicate that Ca²⁺ store depletion stimulates rapid STIM1 surface expression and association with hTRPC1 independently of rises [Ca²⁺]_c. These events require the support of the actin cytoskeleton and are compatible with membrane trafficking underlying de novo coupling of IP₃RII to hTRPC1.