When the β5 (short form) and γ2 subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged αi in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged αi and resulted in purification of β5γ2 to near homogeneity. Subsequent anion-exchange chromatography of β5γ2 resulted in resolution of β5 from γ2 and further purification of β5. The purified β5 eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of γ. β5 monomer could be assembled with partially purified hexahistidine-tagged γ2 in vitro to form a functional dimer that could selectively activate PLC β2 but not PLC β3. αo-GDP inhibited activation of PLC β2 by β5γ2 supporting the idea that β5γ2 can bind to αo. β5 monomer and β5γ2 only supported a small degree of ADP ribosylation of αi by pertussis toxin (PTX), but β5 monomer was able to compete for β1γ2 binding to αi and αo to inhibit PTX-catalyzed ADP ribosylation. These data indicate that β5 functionally interacts with PTX-sensitive GDP α subunits and that β5 subunits can be assembled with γ subunits in vitro to reconstitute activity and also support the idea that there are determinants on β subunits that are selective for even very closely related effectors.