The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtα and Gtβγ subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtγ(60−71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367−373. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin−Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtγ C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtγ for alanine. The mutants were co-expressed with six-histidine-tagged Gtβ subunits in Sf9 insect cells. The Gtβ-6-His-γ mutant proteins were purified and assayed in the presence of Gtα for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*−Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtγ(60−71) region has the most significant effect on rhodopsin−Gt interactions, which places important constraints on the model of the rhodopsin−Gt complex.