The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.