The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Förster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the β₂ adrenoceptor (β₂-AR), a well-characterized GPCR model system. The donor fluorophore, Lumio™Green, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the β₂-AR is 62 Å. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the β₂-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.