The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. In 1995, 2-arachidonylglycerol (2-AG) 1 was isolated from rat brain and canine gut and shown to bind both the central and peripheral cannabinoid receptors (1, 2). Subsequently, 2-AG was shown to be present in vivo at levels several orders of magnitude higher than the other known endocannabinoid, AEA (2-4). Accumulating evidence supports assertions that 2-AG serves as a physiologically relevant cannabinoid receptor ligand, occupying a central role within the endogenous cannabinoid system (5, 6). Therefore, the identification and characterization of enzymes capable of metabolizing this lipid mediator should aid in the elucidation of mechanisms by which cannabinoid tone is modulated in vivo. We are particularly interested in the role of fatty acid oxygenases, such as cyclooxygenases (COXs) and lipoxygenases (LOXs), in 2-AG metabolism and have previously shown that 2-AG is an excellent substrate for COX-2 and leukocyte-type 12-LOX (7, 8).LOXs are a diverse family of nonheme ferroproteins that catalyze the hydroperoxidation of polyunsaturated fatty acids both regio-and stereospecifically (9 -14). Six LOXs have been identified in humans: platelet-type 12-LOX, 12(R)-LOX, 15-LOX-1, 15-LOX-2, e-LOX-3, and 5-LOX (9, 15). The ability of leukocyte 12-LOX, but not platelet 12-LOX, to oxidize 2-AG and the ability of some lipoxygenases to oxidize the endocannabinoid AEA prompted us to evaluate additional possible lipoxygenase metabolic pathways for 2-AG (8, 16 -20). In the present study, we investigated the ability of two plant and four animal LOXs to catalyze the hydroperoxidation of 2-AG. 5-LOX catalyzes the hydroperoxidation of arachidonic acid providing 5-hydroperoxyeicosatetraenoic acid (HpETE), the precursor to the leukotrienes. The possibility that 5-LOX might oxygenate 2-AG to generate 5-HpETE glyceryl ester (HpETE-G) and, subsequently, leukotriene glyceryl esters in a manner similar to the ability of cyclooxygenase-2 to generate prostaglandin glycerol esters was investigated using two 5-LOX enzymes. The endocannabinoid oxygenase activities of four 15-LOX enzymes were also rigorously characterized. Lipoxygenase metabolism of 2-AG in an eukaryotic cellular environment was examined in t...