We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.