Cardiac G protein–coupled receptors that couple to G&agr;sand stimulate cAMP formation (eg, &bgr;-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the &bgr;2-adrenergic receptor (AR), also couples to the inhibitory G protein G&agr;i(activation of which inhibits cAMP formation). If &bgr;2ARs could be shown to couple to G&agr;iin the human heart, it would have important ramifications, because levels of G&agr;iincrease with age and in failing human heart. Therefore, we investigated whether &bgr;2ARs in the human heart activate G&agr;i. By photoaffinity labeling human atrial membranes with [32P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for G&agr;i, we found that G&agr;iis activated by stimulation of &bgr;2ARs but not of &bgr;1ARs. In addition, we found that other G&agr;s-coupled receptors also couple to G&agr;i, including histamine, serotonin, and glucagon. When coupling of these receptors to G&agr;iis disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that &bgr;2AR and many other G&agr;s-coupled receptors in human atrium also couple to G&agr;iand that abolishing the coupling of these receptors to G&agr;iincreases the receptor-mediated adenylyl cyclase activity.