We have applied multicolor BiFC to study the association preferences of G protein beta and gamma subunits in living cells. Cells co-express multiple isoforms of beta and gamma subunits, most of which can form complexes. Although many betagamma complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate betagamma complex formation with functionality in intact cells by comparing the amounts of fluorescent betagamma complexes with their abilities to modulate effector proteins. The relative predominance of specific betagamma complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of beta and gamma subunits by simultaneously visualizing the two fluorescent complexes formed when beta or gamma subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common gamma or beta subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.