Previous mutation and modeling studies have identified an aromatic cluster in the transmembrane helix (TMH) 3-4-5 region as important for ligand binding at the CB(1) and CB(2) cannabinoid receptors. Through novel mixed mode Monte Carlo/Stochastic Dynamics (MC/SD) calculations, we tested the importance of aromaticity at position 5.39(275) in CB(1). MC/SD calculations were performed on wild-type (WT) CB(1) and two mutants, Y5.39(275)F and Y5.39(275)I. Results indicated that while the CB(1) Y5.39(275)F mutant is very similar to WT, the Y5.39(275)I mutant shows pronounced topology changes in the TMH 3-4-5 region. Site-directed mutagenesis studies of tyrosine 5.39 to phenylalanine (Y-->F) or isoleucine (Y-->I) in both CB(1) and CB(2) were performed to determine the functional role of this amino acid in each receptor subtype. HEK 293 cells transfected with mutant receptor cDNAs were evaluated in radioligand binding and cyclic AMP assays. The CB(1) mutant and WT receptors were also co-expressed with G-protein-coupled inwardly rectifying channels (GIRK1 and GIRK4) in Xenopus oocytes to assess functional coupling. The Y-->F mutation resulted in cannnabinoid receptors with subtle differences in WT binding and signal transduction. In contrast, the Y-->I mutations produced receptors that could not produce signal transduction or bind to multiple cannabinoid compounds. However, immunofluorescence data indicate that the Y-->I mutation was compartmentalized and expressed at a level similar to that of the WT cannabinoid receptor. These results underscore the importance of aromaticity at position CB(1) 5.39(275) and CB(2) 5.39(191) for ligand recognition in the cannabinoid receptors.