Guanine nucleotide-binding protein β and γ subunits form a tightly bound complex that can only be separated by denaturation. Assembly of β and γ subunits is a complicated process. The β1 and γ2 subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but β1 cannot form βγ dimers when synthesized in a wheat germ extract. In contrast, γ2 translated in either system can dimerize with β1, suggesting that dimerization-competent γ2 can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent γ2 in solution forms an asymmetric particle with a Stokes radius of about 21 ± 0.4 Å (n = 4), s20,w of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration). To define the part of γ2 that is needed for native βγ dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with β1. Dimerization was assessed by stabilization of β1 to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with β1, whereas removal of 27 amino acids prevented it. Therefore, a region between residues 45 and 59 of γ2 is important for dimerization. Truncation of 15 amino acids from the N terminus greatly diminished the formation of βγ dimers, while removal of 25 amino acids entirely blocked it. Thus, another region important for forming native βγ is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent βγ dimerization. Furthermore, in intact 35S-labeled COS cells, epitope-tagged γ2 coimmunoprecipitates with β and α subunits. The N-terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.