A disulfide bond between two extracellular cysteines, conserved in all G-protein-coupled receptors, is believed to be critical for stabilization of the ligand-binding pocket. The beta 2-adrenergic receptor (beta 2-AR) contains two conserved cysteines (Cys106 and Cys184) as well as two other extracellular cysteines (Cys190 and Cys191). The specificity of the interactions between these four cysteines has not yet been clearly established. Mutants encoding alanines for specific extracellular cysteines in the beta 2-AR gene were constructed and expressed in COS-1 and Chinese hamster ovary cells. Ala106, Ala184,190,191, and Ala106,184,190,191 mutants displayed low affinity for the beta-antagonist, 125I-cyanopindolol and insensitivity to dithiothreitol (DTT). The Ala106,191 mutant displayed an intermediate affinity and DTT sensitivity. Mutants Ala184, Ala184,190, and Ala184,191 displayed high affinity and DTT sensitivity, indicating that a solvent-accessible disulfide bond(s) is present in these mutant receptors as in the wild-type beta 2-AR. Additionally, thermal stability studies provided evidence that the extracellular disulfide bonds are essential for stabilization of the high affinity state of the receptor. These studies indicate that the covalent linkage between loops 1 and 2 of the beta 2-AR extracellular domains involves the formation of disulfide bonds, uniquely between Cys106 and Cys191, and Cys184 and Cys190, and is, thus, distinct from that of other G-protein-coupled receptors.