The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.