The guanine nucleotide-binding protein transducin consists of alpha, beta, and gamma subunits. Like other heterotrimeric G proteins, transducin couples membrane bound receptors with intracellular effectors. To define the structural domains of the transducin alpha subunit that are important for interaction with membrane, membrane-bound receptor and betagamma subunit, Gtalpha was modified by digestion with trypsin, pertussis toxin catalyzed ADP-ribosylation, and truncation at the carboxy terminus. The modified proteins were analyzed in a Triton X-114 phase partitioning system. This detergent, when warmed to 30 degrees C separates into detergent-rich and detergent-depleted phases, with hydrophobic proteins in the detergent-rich phase and soluble proteins in the detergent-depleted phase. As expected, rhodopsin and the farnesylated betagamma subunits partitioned into the hydrophobic phase. Another rod outer segment protein, the 48 kDa arrestin, was hydrophilic. Gtalpha partitioned approximately equally between aqueous and detergent phases. Gtbetagamma subunits caused Gtalpha to behave in a more hydrophobic manner reflecting the interaction between Gtalpha and Gtbetagamma. Rhodopsin alone did not alter the behavior of Gtalpha. Modification of Gtalpha by pertussis toxin-catalyzed ADP-ribosylation appeared not to alter its interaction with Gtbetagamma. The domain of Gtalpha mainly responsible for both hydrophobicity and Gtbetagamma interaction appears to be the amino terminal 2 kDa peptide. Proteolytic removal of this peptide with trypsin resulted in a 37 kDa molecule that partitioned mostly in the aqueous phase both with or without Gtbetagamma. The 20-amino acid carboxyl terminus also appears to impart some degree of hydrophobicity to Gtalpha as recombinant Gtalpha lacking these 20 residues behaved in a more hydrophilic manner than the intact molecule expressed as a recombinant protein.