The endocannabinoid system is a biosignaling network present in mammalian brain and peripheral tissues that includes two druggable G-protein coupled receptors, cannabinoid receptors 1 and 2 (CB1 and CB2). Ligand-assisted protein structure (LAPS) is an experimental method developed at the Center for Drug Discovery (CDD) that integrates molecular biology, proteomics, and molecular modeling to study protein (enzyme, receptor) structure. We have utilized LAPS to determine the ligand-binding and functional domains of CB1 and CB2. æNitrate (ONO2) ester-containing cannabinoids are a new series of compounds developed in CDD. We determined whether compounds bearing the nitrate ester moiety interact covalently with CB-receptor amino acids in a manner that would make these novel cannabinoids useful as site-directed probes for elucidating CB-receptor ligand-binding motif(s). æOne such nitrate ester, AM10209, functions similarly to AM1336, a covalent affinity probe featuring an isothiocyanate moiety. We previously reported that AM1336 binds covalently to both cysteines on helix 7 of hCB2 equivalently and acts pharmacologically as an antagonist/inverse agonist. In the present study we show that AM10209 featuring the nitrate ester moiety attaches covalently to both cysteines on helix 7 equivalently. Thus, the nitrate-ester probe recapitulates the importance of helix-7 cysteine residues in the molecular pharmacology of hCB2 antagonists/inverse agonists as potential medications. æThis work was supported by NIH/NIDA Grants DA003801, DA007215 and DA009158 (to Dr. Makriyannis).