Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A2 (iPLA2s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca2+ signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA2 family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2- one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA2β -/- mice to demonstrate that iPLA2β is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [ 3H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA2β-/- mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA2β-/- mice displayed defects in SMC Ca2+ homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca2+-store depletion resulted in the rapid entry of external Ca2+ into wild-type aortic SMCs that was significantly slower in iPLA2β- null cells (p < 0.01). Furthermore, SMCs from iPLA2β-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA2β-null SMCs were restored by 2 μM AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMCmigration and proliferation in iPLA2β-/- mice. Moreover, PGE2 alone rescued proliferation and migration in iPLA2β-/- mice. We conclude that iPLA2β is an important mediator of AA release and prostaglandin E2 production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.