The G protein betagamma subunit dimer (Gbetagamma) and the Gbeta5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gbetagamma, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gbeta and mediate its interaction with Ggamma. However, it is not known what fraction of the many Gbetagamma combinations is assembled this way or whether chaperones influence the specificity of Gbetagamma dimer formation. Moreover, the mechanism of Gbeta5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Ggamma2 with all four Gbeta1-4 subunits and in the assembly of Gbeta2 with all twelve Ggamma subunits, without affecting the specificity of the Gbetagamma interactions. The results also show that Gbeta5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gbetagamma. PhLP1 seems to stabilize the interaction of Gbeta5 with CCT until Gbeta5 is folded, after which it is released to allow Gbeta5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gbetagamma combinations and suggest a CCT-dependent mechanism for Gbeta5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.