Stimulation of β₂-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of β₂-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na⁺/H⁺ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation^,,,,,. We report here a direct agonist-promoted association of the β₂-adrenergic receptor with the Na⁺/H⁺ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na⁺/H⁺ exchanger type 3 (NHE3). NHERF binds to the β₂-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation ofthe final residue of the β₂-adrenergic receptor from leucine toalanine abolishes the receptor's interaction with NHERF andalso markedly alters β₂-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent β₂-adrenergic receptor binding of NHERF plays a role in β₂-adrenergic receptor-mediated regulation of Na⁺/H⁺ exchange.