Purified mitochondria from germinating castor bean (Ricinus communis L.) endosperm was treated with hydrogen peroxide (H2O2), active oxygen form, in order to investigate the extent of membrane degradation. Incubation of mitochondria with micromolar concentrations (50-200ulcornerM) of H2O2 resulted in a concentraton-dependent loss of membrane proteins. During this process extensive loss of lipid-phosphate content was also observed in mitochondrial membranes. When L-3-phosphatidyl[2-14C]ethanolamine was added to the mitochondrial membranes as an exogenous substrate, the level of radioactivity in the water-soluble fraction was markedly enhanced with increasing concentration of H2O2. Analysis of the water-soluble products formed during the metabolism of ethanolamine-labelled phosphatidylethanolamine by mitochondrial membranes from castor bean indicates that this loss of lipid-phosphate is attributable to action of phospholipase D. Direct measurement of mitochondrial phospholipase D indicated that the activity of enzyme was remarkably stimulated by calcium ion or sodium dodecylsulfate (SDS).The optimum concentrations for enzyme stimulation were 25 and 0.5 mM for calcium ion and SDS in the reaction mixture, respectively. The substrate specificity of phospholipase D was determined by comparing various classes of exogenous phospholoipids, added in the form of sonicated vesicles, as substrates. The phospholipase D exhibited preference for phosphatidylethanolamine. Taken together, our results suggest that increase of mitochondrial phospholipase D activity may be a key event leading to accelerated membrane deterioration following active oxygen attack.