Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.