Previous studies have demonstrated that U937 cells are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in response to receptor-directed and soluble stimuli by a mechanism that involves the activation of Group IV cytosolic phospholipase A(2)alpha. In this paper we show that these cells also mobilize AA in response to an oxidative stress induced by H(2)O(2) through a mechanism that appears not to be mediated by cytosolic phospholipase A(2)alpha but by the calcium-independent Group VI phospholipase A(2) (iPLA(2)). This is supported by the following lines of evidence: (i) the response is essentially calcium-independent, (ii) it is inhibited by bromoenol lactone, and (iii) it is inhibited by an iPLA(2) antisense oligonucleotide. Enzyme assays conducted under a variety of conditions reveal that the specific activity of the iPLA(2) does not change as a result of H(2)O(2) exposure, which argues against the activation of a specific signaling cascade ending in the iPLA(2). Rather, the oxidant acts to perturb membrane homeostasis in a way that the enzyme susceptibility/accessibility to its substrate increases, and this results in altered fatty acid release. In support of this view, not only AA, but also other fatty acids, were found to be liberated in an iPLA(2)-dependent manner in the H(2)O(2)-treated cells. Collectively, these studies underscore the importance of the iPLA(2) in modulating homeostatic fatty acid deacylation reactions and document a potentially important route under pathophysiological conditions for increasing free fatty acid levels during oxidative stress.