Diacylglycerol lipase alpha is the key enzyme in the formation of the most prevalent endocannabinoid, -2arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase alpha, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase alpha, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase alpha. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase alpha by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections. (C) 2011 Elsevier Inc. All rights reserved.