Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of alpha, beta, and gamma subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of alpha from the beta gamma dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the beta gamma dimer or about the areas of beta gamma that interact with alpha. Direct information on the orientation of protein surfaces can be obtained from the analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links alpha to beta and beta to gamma (Yi, F., Denker, B. M., and Neer, E. J. (1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in beta and gamma involved in cross-linking to each other or to alpha, we have mutated the cysteines in beta 1, gamma 2, and gamma 3 and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form beta gamma dimers that could interact with the alpha subunit. We found that 1,6-bismaleimidohexane can cross-link beta 1 to gamma 3 but not to gamma 2. The cross-link goes from Cys25 in beta 1 to Cys30 in gamma 3. This cysteine is absent from any of the other known gamma isoforms and therefore confers a distinctive property to gamma 3. The beta subunit in the beta 1 gamma 2 dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked beta 1 gamma 3. The beta subunit can also be cross-linked to alpha, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking beta to Cys215 in alpha o (Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10295-10299). Mutation of Cys204 in beta 1 abolished one of these two products, whereas mutation of Cys271 abolished the other. Because both alpha-beta cross-linked products are formed in approximately equal amounts, Cys204 and Cys271 in beta are equally accessible from Cys215 in alpha o. Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the beta gamma dimer.