Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ entry in human platelets, although the mechanism of SOCE activation has not been clearly established. In one model, Ca²⁺ store depletion induces de novo conformational coupling of the type II IP₃ receptor to hTRPC1, believed to be a component of the store-operated channel. This coupling requires basal levels of IP₃ and functional IP₃ receptors (1, 2). In contrast, an alternative model suggests that Ca²⁺ store depletion induces formation of a Ca²⁺ influx factor (CIF) that displaces calmodulin (CaM) from Ca²⁺-independent phospholipase A₂ (iPLA₂), resulting in iPLA₂ activation and lysophospholipid production, which then activates the store-operated channel (3). Here the role of iPLA₂ in SOCE activation in human platelets has been investigated. Fura-2–loaded human platelets were stimulated with the CaM inhibitor, calmidazolium (CMZ), resulting in a concentration-dependent increase in Ca²⁺ release and Ca²⁺ entry. At no concentration was Ca²⁺ entry seen without Ca²⁺ release. No Ca²⁺ entry was detected using 1 μM CMZ, and only a relatively small Ca²⁺ entry was detected using 10 μM. In contrast, iPLA₂ was strongly activated by 1 μM CMZ, and maximally activated by 10 μM. These data suggest that iPLA₂ activation in not sufficient for SOCE activation in these cells. Thapsigargin-evoked SOCE was completely abolished by prior incubation with the iPLA2 inhibitor, bromoenol lactone (BEL; 25 μM; 30 min). BEL was found to increase resting intracellular Ca²⁺ concentration from 61 ± 4 nM to 155 ± 2 nM (mean ± SEM; n = 6; p < 0.005; Student’s t test). BEL also reduced PAR-1 –dependent Ca²⁺ release to 20.2 ± 2.1% of control (n = 6; p < 0.005), which appeared to be due to inhibition of agonist-evoked IP₃ synthesis. IP₃ levels in cells stimulated by the PAR-1 agonist, SFLLRN, after BEL treatment were not significantly higher than basal (100.9 ± 10.2% of control; n = 6; p = 0.48), as measured by a fluorescence polarisation assay. Inhibition of phospholipase C by Et-18-OCH₃ also significantly inhibited TG-evoked SOCE to 12.7 ± 3.6% of control (n = 9; p < 0.001). This is consistent with an essential role for basal IP₃ in the activation of SOCE (1). The disruption of IP₃ production by BEL may be owing to the role of iPLA₂ in remodelling of the fatty-acid content of membrane phospholipids. In conclusion, although iPLA₂ appears to have an important role in regulation of cell signalling through the remodelling of cellular phospholipids, it is unlikely to play a direct role in SOCE activation in human platelets.