The cytoplasmic surface of G protein-coupled receptors plays a central role for activation and deactivation of the receptor. To understand the molecular mechanisms which underlie these processes, we determined the surface charge density and its changes upon activation directly at the cytoplasmic surface of bovine rhodopsin and correlated these changes with key events in receptor activation. The surface charge density was calculated from the ionic strength dependence of the apparent pKa of the surface-bound pH-indicator dye fluorescein according to the Gouy-Chapman theory. The surface charge density at pH 6.5 changes by 0.8 ± 0.2 elementary charge/1000 Å2 in rod outer segment disk membranes and by 0.4 ± 0.2 elementary charge/1000 Å2 in rhodopsin/dodecylmaltoside micelles upon formation of the active metarhodopsin-II state. By comparison of these surface charge density values determined with and without the native lipid environment, we calculated the charge change to about 1 elementary charge/cytoplasmic rhodopsin surface. The more positive surface charge density in metarhodopsin-II decreases back to the dark state level of σ = −2.0 ± 0.2 elementary charges/1000 Å2 in the opsin state, providing further evidence that the cytoplasmic surface properties after metarhodopsin-II decay resemble almost those of the dark state.