Cysteine351 is the site for pertussis toxin-catalyzed ADP-ribosylation in the G protein Gi1 alpha. Alteration of this residue, or the equivalent cysteine in other Gi-family G proteins, has been used to examine specific interactions between receptors and these G proteins. However, no systematic analysis has been performed to determine the quantitative effect of such alterations. To address this we mutated cysteine351 of Gi1 alpha to all other possible amino acids. Each of the G protein mutants was transiently coexpressed along with the porcine alpha 2A-adrenoceptor in HEK 293/T cells. Following pertussis toxin treatment of the cells, membranes were prepared and the capacity of the agonist UK14304 to stimulate the binding of [35S]GTP gamma S to the modified G proteins was measured. A spectrum of function was observed. The presence of either a charged amino acid or a proline at this position essentially attenuated agonist regulation. The wild-type G protein did not result in maximal stimulation by agonist. The presence of certain branched chain aliphatic amino acids or bulky aromatic R groups at amino acid351 resulted in substantially greater maximal stimulation by the alpha 2A-adrenoceptor than that achieved with the wild-type sequence. The degree of activation of the forms of Gi1 alpha correlated strongly with the octanol/water partition coefficient of the amino acid at residue351. Variation in EC50 values for agonist-induced stimulation of binding of [35S]GTP gamma S to the mutant G proteins also correlated with the octanol/water partition coefficient. These results define a central role for hydrophobicity of this residue in defining productive receptor-G protein interactions.