The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.