Cytosolic phospholipase A2 (cPLA2) selectively catalyses the release of arachidonic acid from the sn-2 position of glycero-phospholipids to produce prostaglandins and leukotrienes. In this study, vitamin E enrichment of rat heart myoblastic H9c2 cells caused an increase in the release of arachidonate during ionophore (A23187) stimulation. PLA2 activity in the cytosolic fraction was also enhanced but enzyme activity in the particulate fraction was not affected by this treatment. Immunoblotting analysis with a polyclonal anti-cPLA2 antibody showed an increased level of the enzyme in vitamin E-treated cells. Direct incorporation of vitamin E into lipid vesicles in the assay mixture resulted in modulation of enzyme activity in a biphasic manner. Pretreatment of cells with phorbol 12-myristate 13-acetate, a known activator of protein kinase C, synergistically potentiated the ionophore-induced arachidonate release in both the control and vitamin E-treated cells. However, vitamin E treatment by itself did not affect the protein kinase C activity, indicating that the vitamin E-induced activation of cPLA2 was independent of the protein kinase C cascade. Collectively, these results suggest that vitamin E potentiates arachidonate release through the direct and/or indirect modulation of cPLA2 activity.