Heterotrimeric G proteins regulate multiple effectors of which some are mediated via the Gbetagamma dimers. There is evidence to suggest that the functions of Gbetagamma dimers are not shared by all possible permutations of Gbetagamma complexes. Here, we report our efforts in defining the formation of distinct Gbetagamma dimers and their functional differences in activating phospholipase Cbeta (PLCbeta) isoforms. Co-immunoprecipitation assays using Cos-7 cells transiently expressing 48 different combinations of Gbeta(1-4) and Ggamma(1-5, 7-13) subunits showed that Gbeta(1) and Gbeta(4) could form dimers with all known Ggamma subunits, whereas several dimers could not be observed for Gbeta(2) and Gbeta(3). All Gbeta(1)gamma and Gbeta(2)gamma dimers significantly stimulated PLCbeta isoforms (PLCbeta(2)> or =PLCbeta(3)>PLCbeta(1)), but Gbeta(3)gamma and Gbeta(4)gamma dimers were poor activators of PLCbeta(1) and exhibited preference for PLCbeta(3) and PLCbeta(2), respectively. All Gbeta subunits revealed to date contain the previously identified PLCbeta(2)-interacting residues, but their neighboring residues in the proposed 3-D structures are different. To test if differences in these neighboring residues affect the interactions with PLCbeta isoforms, we generated several Gbeta(3) mutants by replacing one or more of these residues with their Gbeta(1) counterparts. One of these mutants (M120I, S140A and A141G triple mutant) acquired enhanced PLCbeta(2)-activating functions when co-expressed with different Ggamma subunits, while the corresponding stimulation on PLCbeta(3) was not altered. Taken together, our results show that the exact composition of a Gbetagamma dimer can determine its selectivity for activating PLCbeta isoforms, and certain residues in Gbeta(3) may account for the preferential stimulation of PLCbeta(3) by Gbeta(3)gamma dimers.