Activation of the G protein-coupled receptor rhodopsin involves both the motion of transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled rhodopsin in solutions of dodecyl maltoside was used so that time-resolved TM6 motion and proton exchange could each be monitored as a function of pH and temperature after an activating light flash. The results reveal that the motion of TM6 is not synchronized with deprotonation of the Schiff base that binds the chromophore to the protein but is an order of magnitude slower at 30 degrees C. However, TM6 motion and the uptake of a proton from solution in the neutral pH range follow the same time course. Importantly, the motion of TM6 is virtually independent of pH, as is Schiff base deprotonation under the conditions used, whereas proton uptake titrates with a pK of 6.5. This finding shows that proton uptake is a consequence rather than a cause of helix motion. Activated rhodopsin binds to and subsequently activates the cognate G protein, transducin. It has been shown that peptides derived from the C terminus of the transducin alpha-subunit mimic in part binding of the intact G protein. These peptides are found to bind to rhodopsin after TM6 movement, resulting in the release of protons. Collectively, the data suggest the following temporal sequence of events involved in activation: (i) internal Schiff base proton transfer; (ii) TM6 movement; and (iii) proton uptake from solution and binding of transducin.