Cytosolic phospholipase A2 (cPLA2) is a signal-responsive enzyme that is highly selective to the nature of phospholipid substrates. A mechanism for cPLA2 activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants. Oxidant-mediated signaling of PLA2 may serve as an alternative mechanism for enzyme regulation; however, the manner of regulation has yet to be delineated. In this report we demonstrate that there is a direct effect of membrane oxidation on cPLA2 phosphorylation and activity. A simple in vitro system consisting of purified cPLA2 and phospholipid vesicles was used to facilitate protein kinase C (PKC) activity and provide substrates for cPLA2. Using these vesicles we found that the activity of cPLA2 was enhanced twofold when the vesicles contained as little as 5 mol% phosphatidylcholine hydroperoxides (PLPCOOH). The order of hydrolytic preference for fatty acyl species was 20:4 > 18:2 > 18:1 > 16:0, and the presence of PLPCOOH stimulated hydrolysis largely of phosphatidylcholine containing 20:4. The Ca2+ concentrations required for stimulated hydrolytic activity were also twofold lower for oxidized compared to unoxidized vesicles. Using phospholipid micelles as substrates, PKC-mediated phosphorylation of cPLA2 increased hydrolytic activity 71% compared to preparations lacking PKC. Using phospholipid vesicles as substrates, PKC-mediated phosphorylation resulted in an 85% increase in cPLA2 activity compared to preparations without PKC. PKC-mediated phosphorylation of cPLA2, therefore, stimulates catalytic activity toward membrane phospholipids and the extent of activation is enhanced directly by peroxidation of membrane phospholipids and involves a peroxide-induced stimulation of cPLA2 phosphorylation.