Some G-protein-coupled receptors regulate biological processes via Gα12/13- or Gαq/11-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF, we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or muscarinic M3 receptors, a robust FRET signal occurred that allowed for the first time the analysis of the kinetics of this interaction in detail. On- and off-set kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of Gαq activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins and dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore, we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF-mediated downstream signalling in the presence of RGS2. In summary, these observations support the concept of a functional activation-dependent p63RhoGEF-Gαq-RGS2 complex.