Dynamic regulation of G-protein signaling in the phototransduction cascade ensures the high temporal resolution of vision. In a key step, the activated alpha-subunit of transducin (Galphat-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory gamma-subunit (PDEgamma). Significant progress in understanding the interaction between Galphat and PDEgamma was achieved by solving the crystal structure of the PDEgamma C-terminal peptide bound to Galphat in the transition state for GTP hydrolysis (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Nature 409, 1071-1077). However, some of the structural elements of each molecule were absent in the crystal structure. We have probed the binding surface between the PDEgamma C terminus and activated Galphat bound to guanosine 5'-O-(3-thio)-triphosphate (GTPgammaS) using a series of full-length PDEgamma photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDEgamma photoprobe species, expressed protein ligation allowed one benzoyl-L-phenylalaine substitution at selected hydrophobic C-terminal positions, and the addition of a biotin affinity tag at the extreme C terminus. We have detected photocross-linking from several PDEgamma C-terminal positions to the Galphat-GTPgammaS N terminus, particularly from PDEgamma residue 73. The overall percentage of cross-linking to the Galphat-GTPgammaSN terminus was analyzed using a far Western method for examining Galphat-GTPgammaS proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to Galphat from a benzoyl-phenylalanine replacement at PDEgamma position 86 localized the region of photoinsertion to Galphat N-terminal residues Galphat-(22-26). This novel Galphat/PDEgamma interaction suggests that the transducin N terminus plays an active role in signal transduction.