The humanmuopioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr- D-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoidCB1receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoidCB1agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [35S]guanosine 5 -3-O-(thio)triphosphate (GTP S) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [35S]GTP S binding. CB1 inverse agonists attenuated basal [35S]GTP S binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of coexpressed mu opioid receptor to function.