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The mitochondrial calcium independent phospholipase 2γ (iPLA2γ) catalyzes the hydrolysis of membrane glycerophospholipids with subsequent release of free fatty acids and lysophospholipids. The activity of iPLA2γ is upregulated by H 2 O in isolated mitochondria and reconstituted system, suggesting activation by redox modification of accessible cysteine residues. Here we tested the hypothesis that iPLA2γ is activated also by lipid hydroperoxides. We treated purified recombinant iPLA2γ with cardiolipin hydroperoxides (CLOOHs) or dithiothreitol, together with approaches for the detection of sulfenic acid modified proteins. We found that CLOOH treatment resulted in oxidation of iPLA2γ, as evaluated from the different electrophoretic mobilities of the oxidized and reduced forms. In addition, measurements of recombinant iPLA2γ activity showed that the Vmax values obtained with CLOOH as the substrate were identical to the values obtained with H 2 O 2 -activated iPLA2γ and cardiolipin as the substrate. These results indicate that lipid hydroperoxides participate in the regulation of iPLA2γ by reversible oxidation of accessible cysteine residues and identify CLOOHs as both substrates and redox activators of the enzyme. [ABSTRACT FROM AUTHOR]