G protein-coupled receptors play a central role in signal transduction and were only known to be activated by agonists. Recently it has been shown that membrane potential also affects the activity of G protein-coupled receptors. For the M(2) muscarinic receptor, it was further shown that depolarization induces charge movement. A tight correlation was found between the voltage-dependence of the charge movement and the voltage-dependence of the agonist binding. Here we examine whether depolarization-induced charge movement causes a conformational change in the M(2) receptor that may be responsible for the voltage-dependence of agonist binding. Using site-directed fluorescence labeling we show a voltage-dependent fluorescence signal, reflecting a conformational change, which correlates with the voltage-dependent charge movement. We further show that selected mutations in the orthosteric site abolish the fluorescence signal and concomitantly, the voltage-dependence of the agonist binding. Surprisingly, mutations in the allosteric site also abolished the voltage-dependence of agonist binding but did not reduce the fluorescence signal. Finally, we show that treatments, which reduced the charge movement or hindered the coupling between the charge movement and the voltage-dependent binding, also reduced the fluorescence signal. Our results demonstrate that depolarization-induced conformational changes in the orthosteric binding site underlie the voltage-dependence of agonist binding. Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltage-dependent agonist binding.