To analyze individual steps of G(S)-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G(S) activation in living cells. This was done by coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gbetagamma subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G(S) signaling cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction was on the same time scale as A(2A) receptor activation with a time constant of <50 ms. G(S) activation was markedly slower and around 450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors. Taken together, our kinetic measurements demonstrate that the rate of G(S) activation limits initiation of G(S)-coupled receptor signaling.