The cannabinoid CB(1) (CB(1)) and dopamine D(2) (D(2)) receptors are coexpressed in the basal ganglia, an area of the brain involved in such processes as cognition, motor function, and emotional control. Several lines of evidence suggest that CB(1) and D(2) receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB(1) and D(2) receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB(1)-D(2L) heterodimers as well as D(2L)-D(2L) homodimers in a neuronal cell model, Cath. a differentiated cells. MBiFC was then used to explore the effects of persistent ligand treatment on receptor dimerization at the plasma membrane and intracellularly. Persistent (20-h) agonist treatment resulted in increased formation of CB(1)-D(2L) heterodimers relative to the D(2L)-D(2L) homodimers. The effects of the D(2) agonist quinpirole were restricted to the intracellular compartment and may reflect increased D(2L) receptor expression. In contrast, treatment with the CB(1) receptor agonist (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55, 940) produced increases in both membrane and intracellular CB(1)-D(2L) heterodimers independently of alterations in CB(1) receptor expression. The effects of CB(1) receptor activation were attenuated by the CB(1) antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both time- and dose-dependent. The effects of CB(1) activation were examined further by combining MBiFC with a constitutively active CB(1) receptor mutant, CB(1)T210I. These studies demonstrated that the expression of CB(1)T210I increased intracellular CB(1)-D(2L) heterodimer formation. In summary, agonist-induced modulation of CB(1)-D(2L) oligomerization may have physiological implications in diseases such as Parkinson's disease and drug abuse.