Photoactivated rhodopsin (R*) catalyzes nucleotide exchange by transducin, the heterotrimeric G protein of the rod cell. Recently, we showed that certain alanine replacement mutants of the α5 helix of the α subunit of transducin (Gα_t) displayed very rapid nucleotide exchange rates even in the absence of R* [Marin, E. P., Krishna, A. G., and Sakmar, T. P. (2001) J. Biol. Chem. 276, 27400−27405]. We suggested that R* catalyzes nucleotide exchange by perturbing residues on the α5 helix. Here, we characterize deletion, insertion, and proline replacement mutants of amino acid residues in α5. In general, the proline mutants exhibited rates of uncatalyzed nucleotide exchange that were 4−8-fold greater than wild type. The proline mutants also generally displayed decreased rates of R*-catalyzed activation. The degree of reduction of the activation rate correlated with the position of the residue replaced with proline. Mutants with replacement of residues at the amino terminus of α5 exhibited mild (<2-fold) decreases, whereas mutants with replacement of residues at the carboxyl terminus of α5 were completely resistant to R*-catalyzed activation. In addition, insertion of a single helical turn in the form of four alanine residues following Ile339 at the carboxyl terminus of α5 prevented R*-catalyzed activation. Together, the results provide evidence that α5 serves an important function in mediating R*-catalyzed nucleotide exchange. In particular, the data suggest the importance of the connection between the α5 helix and the adjacent carboxyl-terminal region of Gα_t.