G proteins act as molecular switches in which information flow depends on whether the bound nucleotide is GDP ("off") or GTP ("on"). We studied the basal and receptor-catalyzed nucleotide exchange rates of site-directed mutants of the α subunit of transducin. We identified three amino acid residues (Thr-325, Val-328, and Phe-332) in which mutation resulted in dramatic increases (up to 165-fold) in basal nucleotide exchange rates in addition to enhanced receptor-catalyzed nucleotide exchange rates. These three residues are located on the inward facing surface of the α5 helix, which lies between the carboxyl-terminal tail and a loop contacting the nucleotide-binding pocket. Mutation of amino acid residues on the outward facing surface of the same α5 helix caused a decrease in receptor-catalyzed nucleotide exchange. We propose that the α5 helix comprises a functional microdomain in G proteins that affects basal nucleotide release rates and mediates receptor-catalyzed nucleotide exchange at a distance from the nucleotide-binding pocket.