Store-operated Ca²⁺ entry by de novo conformational coupling between hTRPC1 channels in the plasma membrane and type II IP₃ receptors in the Ca²⁺ stores has been presented as a major pathway for Ca²⁺ entry in human platelets. The cytochrome P450 metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET) induces divalent cation entry sensitive to 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with the anti-hTRPC1 antibody. Ca²⁺ entry stimulated by low concentrations of thapsigargin, which selectively deplete the dense tubular system, was blocked by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE-mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. 5,6-EET-induced Ca²⁺ entry requires basal levels of H₂O₂. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca²⁺ store towards the plasma membrane to facilitate de novo coupling between IP₃ receptor type II and hTRPC1. Supported by MEC-DGI grant BFU2004-00165 and Ministerio de Asuntos Exteriores y Cooperación de España (38/04/P/E).