Endocannabinoids are lipid-signaling molecules that bind cannabinoid receptors. These G-protein-coupled receptors were discovered as binding sites for Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa) (1). Two types of cannabinoid receptors are known: cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) (2,3). CB1 is expressed in the central and peripheral nervous system. CB2 is expressed in immune cells such as macrophages and B lymphocytes (4). 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid found in the brain. 2-AG plays an important role in a variety of physiological functions including neuroprotection, regulation of food intake, anti-inflammation, and anti-nociception (4,5). It is believed that 2-AG is produced on demand in post-synaptic neurons, secreted, and acts at CB1 receptors expressed on pre-synaptic neurons (4). Monoacylglycerol lipase (MAGL) is co-expressed with CB1 receptors and is responsible for ∼85% of 2-AG hydrolysis in the brain (6–8). MAGL is a 33-kDa serine hydrolase containing a catalytic triad (Ser-122, Asp-239, and His-269 in the mouse homolog) characteristic of members of this superfamily of proteins (9). 2-AG hydrolysis may be quantified by mass spectrometry or assays with radioactive substrate. Mass spectrometry is impractical for high-throughput assays, while radiolabeled 2-AG is very costly. Radiolabeled 2-oleoylglycerol (2-OG), which does not bind to cannabinoid receptors, is often used as a substitute for 2-AG since MAGL hydrolyzes both at similar rates (10,11). Nevertheless, radioactive 2-OG is still relatively expensive. In this study, we explored an alternative method of measuring 2-AG hydrolysis based upon reagents originally developed by Cayman Chemical Company to measure the potency of MAGL inhibitors. The assay employs a thioester-containing analog of 2-AG, arachidonoyl-1-thio-glycerol (A-1-TG), which MAGL can hydrolyze. The thioglycerol released upon hydrolysis by MAGL reacts with 5,5′-dithiobis(2-dinitrobenzoic acid) (DTNB) resulting in the release of a yellow thiolate ion (TNB) (Fig. 1) (12). This assay was utilized to measure MAGL kinetics, activity in cytosolic and membrane fractions, and loss of activity in a catalytically inactive mutant. Saario et al. developed the inhibitor N-arachidonyl maleimide (NAM) that irreversibly binds to sulfhydryl groups of cysteine residues (Cys208 and Cys242) in the active site of MAGL (13–15). The potency of this inhibitor was measured using the spectrophotometric assay and a conventional radioactive assay using 3H-2-OG for comparison. These experiments serve to measure the assay’s applicability and limitations. Fig. 1 Scheme for arachidonoyl-1-thio-glycerol hydrolysis and the subsequent color reaction. Arachidonoyl-1-thio-glycerol is hydrolyzed by MAGL releasing a thioglycerol. The thioglycerol reacts with DTNB releasing the yellow-colored ion, TNB MATERIALS AND METHODS Materials N-arachidonyl maleimide, methylarachidonyl fluorophosphonate, and arachidonoyl-1-thio-glycerol were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). 5,5′-dithiobis(2-dinitrobenzoic acid) and 2-oleoylglycerol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mono-oleoyl glycerol racemic 2-[glycerol-1 2 3-3H] (1 mCi/mL) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA).