Reactive oxygen species (ROS) have been implicated in the pathogenesis of diseases as well as various normal cellular processes. It has been suggested that ROS function as mediators of signal transduction, given that they can mimic growth factor-induced signaling. The ROS H2O2 has been reported to activate phospholipase A2 (PLA2) and, therefore, we investigated if and through which pathway ROS activate cytosolic PLA2 (cPLA2) in Her14 fibroblasts. cPLA2 was activated concentration-dependently by H2O2 in a transient manner. In addition, the lipophilic cumene hydroperoxide was shown to induce cPLA2 activity in the same manner. H2O2-induced cPLA2 activity in Her14 cells was partially phosphorylation-dependent, which was mediated through the Raf-MEK-p42/44(MAPK) pathway and occurred partially through a phosphorylation-independent mechanism. ROS can lead to changes in the (micro) viscosity of membranes due to the presence oxidized lipids, thereby increasing the substrate availability for cPLA2. In support of this, treatment of Her14 cells with H2O2 induced lipid peroxidation time-dependently as determined from degradation of lipid arachidonate and linoleate and the formation of aldehydic degradation products. Furthermore, H2O2 induced translocation of cPLA2 to the membrane fraction in a calcium-independent fashion, with a concomitant increase in cPLA2 activity. Collectively, the results suggest that oxidative stress-induced cPLA2 activity is partially phosphorylation-dependent and is further increased due to increased substrate availability by the action of ROS on membranes.