Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A(2a) receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist- or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17 degrees C and 9 degrees C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.