The human neuronal cannabinoid receptor (CB1) is a G-protein-coupled receptor (GPCR) triggered by the psychoactive ingredients in marijuana, as well as endogenous cannabinoids produced in the brain. As with most GPCRs, the mechanism of CB1 activation is poorly understood. In this work, we have assessed the role of cysteine residues in CB1 ligand binding and activation, and demonstrate a method for mapping key determinants in CB1 structure and function. Through mutational analysis, we find that only two cysteines, C257 and C264, are required for high-level expression and receptor function. In addition, through cysteine reactivity studies, we find that a cysteine in transmembrane helix seven, C386 (C7.42), is reactive toward methanethiosulfonate (MTS) sulfhydryl labeling agents, and is thus solvent accessible. Interestingly, steric bulk introduced at this site, either through MTS labeling or by mutation, inhibits binding of the antagonist drug SR141716A (also known as Rimonabant or Accomplia), but does not affect the binding of the agonist CP55940. Our subsequent modeling studies suggest this effect is caused by steric clash of the modified C386 residue with the piperidine ring of SR141716A and/or disruption of an aromatic microdomain in the binding pocket. On the basis of these results, we hypothesize that bound SR141716A inhibits the ability of transmembrane helix 6 to move during formation of the functionally active receptor state.