To investigate how specific amino acid residues affect human cannabinoid CB1 receptor binding and activation, CHO cell lines stably expressing wild type and the phenylalanine 200 to alanine mutant of human cannabinoid CB1 receptor (F200A) were examined. AM2233 functions as an agonist at the wild type receptor (EC50=0.93 nM), but behaves as an inverse agonist at F200A (EC50=4.8 nM). The F200A mutant has significantly lower forskolin-stimulated basal cAMP accumulation than that of the wild type, indicating that the F200A mutant possesses higher constitutive activity. F200 doesn't contribute substantially to the high affinity binding of AM2233 at human cannabinoid CB1 receptor. CP55940, HU-210 and Win55212-2 still function as agonists at the F200A mutant, with similar efficacy, potency, and apparent binding affinity for both wild type human cannabinoid CB1 receptor and F200A mutant. These data indicate that the phenylalanine 200 residue in human cannabinoid CB1 receptor is involved in the receptor activation induced by a specific class of agonists, and supports a model of agonist-structure-dependent conformational changes.