Herein we present multiple lines of evidence which demonstrate that depletion of internal calcium stores is both necessary and sufficient for the activation of calcium-independent phospholipase A2 during arginine vasopressin (AVP)-mediated mobilization of arachidonic acid in A-10 smooth muscle cells. First, AVP-induced [3H]arachidonic acid release was independent of increases in cytosolic calcium yet was decreased by pharmacological inhibition of the release of calcium ion from internal stores. Second, thapsigargin induced the dramatic release of [3H]arachidonic acid from A-10 cells at a similar rate as the AVP-induced release of arachidonic acid, and the release of arachidonic acid by either AVP or thapsigargin was entirely inhibited by (E)-6-(bromomethylene)-3-(1- naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Third, the magnitude of thapsigargin-induced [3H]arachidonic acid release was entirely independent of alterations in cytosolic calcium concentration. Fourth, A23187 resulted in the BEL-inhibitable release of [3H]arachidonic acid from A-10 cells even when ionophore-induced increases in cytosolic calcium were completely prevented by calcium chelators. Fifth, pretreatment of A-10 cells with a calmodulin antagonist (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl) resulted in the time-dependent decrease of subsequent thapsigargin- induced [3H]arachidonic acid release. Collectively, these results identify a novel paradigm which links alterations in calcium homeostasis to the calmodulin-mediated regulation of calcium-independent phospholipase A2 through the depletion of internal calcium stores.