Store-operated channels (SOC) and Ca²⁺ entry (SOCE) are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, as voltage-gated L-type Ca²⁺ channels (Ca²⁺_L) are thought to be fully responsible for agonist-induced Ca²⁺ influx and vasoconstriction. Here we present evidence that SOC channels and their activation via a Ca²⁺-independent phospholipase A₂ (iPLA₂)-mediated pathway plays a crucial role in agonist-induced constriction of cerebral, mesenteric and carotid arteries. Intracellular Ca²⁺ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that: 1) Ca²⁺ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (Nim, Ca²⁺_Linhibitor), and by 2-aminoethyl diphenylborinate (2-APB, inhibitor of SOC), suggesting that SOC and Ca²⁺_Lboth may be involved in agonist-induced constriction of cerebral arteries; 2) functional inhibition of iPLA₂ totally inhibited PE-induced Ca²⁺ influx and constriction in cerebral, mesenteric and carotid arteries, while K⁺-induced Ca²⁺ influx and vasoconstriction mediated by Ca²⁺_L channels remained intact. Thus, iPLA2-dependent activation of SOC channels is crucial for agonist-induced Ca²⁺ influx and vasoconstriction in cerebral, mesenteric and carotid arteries. We propose that upon PE-induced depletion of Ca²⁺ stores, nonselective SOC channels are activated via iPLA₂-dependent pathway, and may produce depolarization of SMC that could trigger a secondary activation of Ca²⁺_L channels, which lead to Ca²⁺ entry and vasoconstriction