In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the alpha subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTPderivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha3 and alpha4/beta6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha3 helix), Met-308, and Arg-310 (alpha4/beta6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha3 and alpha4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha3/alpha4/beta6 region of GTP-bound transducin alpha subunit.