To better understand the mechanism by which the activating signal is transmitted from the receptor-interacting regions on the G protein α-subunit (Gα) to the guanine nucleotide-binding pocket, we generated and characterized mutant forms of Gα with alterations in switch II (Trp-207→Phe) and the carboxyl-terminus (Phe-350→Ala). Previously reported bacterial expression methods for the high-level production of a uniformly isotope-labeled Gtα/Gi1α chimera, ChiT, were successfully used to isolate milligram quantities of 15N-labeled mutant protein. NMR analysis showed that while the GDP/Mg2+-bound state of both mutants shared an overall conformation similar to that of the GDP/Mg2+-bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF4−) revealed distinct differences between the wild-type and mutant Gα subunits, particularly in the response of the 1HN, 15N cross-peak for the Trp-254 indole in the Trp-207→Phe mutant and the 1HN, 15N cross-peak for Ala-350 in the Phe-350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to Gtα and ChiT upon formation of the “transition/activated” state in the presence of AlF4−, whereas the intrinsic fluorescence of the Trp-207→Phe mutant decreased. These results show that the substitution of key amino acid positions in Gα can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.