Activation of brain mitochondrial phospholipase(s) A(2) (PLA(2)) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca(2+)-dependent PLA(2) having biochemical properties characteristic to secreted PLA(2) (sPLA(2)) and immunoreacting with the antibody raised against recombinant type IIA sPLA(2) (sPLA(2)-IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA(2) from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC(6)(3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA(2) release. The increase of sPLA(2) specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA(2) has mainly nuclear localization. In addition to sPLA(2), mitochondria contain another phospholipase A(2) that is Ca(2+)-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA(2)-IIA that might contribute to cell damage.