Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho*, which binds the heterotrimeric G protein, transducin (G(t)) causing GDP to GTP exchange and G(t) dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1 mM n-dodecyl-beta-D-maltoside (DDM) and Rho monomers in 5 mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho*.G(t) and sConA-Rho*.G(t) complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho*.G(t)-LMNG complexes accommodated two Rho molecules, one G(t) heterotrimer and a detergent belt. Visualization of triple sConA-Rho*.G(t) complexes unequivocally demonstrated a pentameric assembly of the Rho*.G(t) complex in which the photoactivated Rho* dimer serves as a platform for binding the G(t) heterotrimer. Importantly, individual monomers of the Rho* dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal. (c) 2013 Elsevier Inc. All rights reserved.