Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.