We (66) described a non-steroidal anti-inflammatory drug (NSAID) insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis (by calcium independent mitochondrial iPLA2) of oxidized CL (CLox) leading to formation of lyso-CL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAEC) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by Lipoprotein-associated phospholipase A2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species.. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolyso-cardiolipin and dilyso-cardiolipin and oxidized octadecadienoic metabolites including 9- and 13- hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2. Lower doses of non-lethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolyso-cardiolipin or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa infection, 34 +/- 8 molar % (n=6; P<0.02) of circulating CL was oxidized. In addition, molar percent of monolyso-cardiolipin increased by two fold after P. aeruginosa in a subgroup analyzed for these changes. Collectively these studies suggest an important role for: a) oxidation of cardiolipin in pro-inflammatory environments; and b) possible hydrolysis of CLox in extracellular spaces producing lyso-CL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.