Excitatory agonists can induce significant smooth muscle contraction under constant free Ca(2+) through a mechanism called Ca(2+) sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca(2+)-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA(2) activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca(2+) sensitization of contraction from 49 +/- 3.6 to 12 +/- 1.0% (p < 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca(2+), phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA(2) in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA(2) is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca(2+)-sensitization of contraction in vascular smooth muscle.