Previous studies revealed that human fat cell plasma membranes contain a multireceptor-linked H 2 O 2 -generating system that is under antagonistic control by hormones and cytokines and is stimulated by insulin via G␣ i2 . In this report, it is shown that the inhibitory action of the ␤-adrenergic agonist isoproterenol is mediated by G protein ␤␥-subunits, based on observations that its action was specifically reversed by anti-G␤ antibodies or a C-terminal ␤-adrenergic receptor kinase-1 fragment containing the G␤␥-binding site of the enzyme, and was mimicked by exogenously supplied G protein ␤␥-sub- There is growing evidence that redox-active biomolecules play important roles in signaling by hormones and cytokines (1-3). Ligand-induced changes in cellular redox status modulate tyrosine phosphorylation-dependent pathways of signal transduction; alter DNA binding and transactivation activities of many transcriptional activators; and may influence key steps in the synthesis, degradation, and action of cAMP and cGMP as well (2, 3). Previous work showed that human fat cells possess a plasma membrane-bound H 2 O 2 -generating system that is under antagonistic control by a large and diverse group of hormones and cytokines, including insulin, the ␤-adrenergic agonist isoproterenol, and different isoforms of fibroblast and platelet-derived growth factors (4 -7). The mechanisms by which hormones and cytokines regulated NADPH-dependent H 2 O 2 generation were confined to the plasma membrane, operated in the absence of ATP, and were independent of soluble second messengers, indicating that established pathways of signal transduction were not involved. These findings placed human fat cell oxidase in a position comparable with adenylyl cyclase and suggested a physical interaction between receptors and NADPH oxidase or receptor-effector coupling via signaltransducing protein(s). Indeed, recent work revealed that the stimulatory effect of insulin on NADPH-dependent H 2 O 2 generation is transduced via G␣ i2 (7).In this study, we have investigated whether the effects of the ␤-adrenergic agonist isoproterenol are also mediated by heterotrimeric G protein(s). Using specific antibodies against the ␣-and ␤-subunits of heterotrimeric G proteins and a peptide that specifically binds G␤␥, it is shown that the inhibitory effects of isoproterenol on NADPH-dependent H 2 O 2 generation are mediated by ␤␥-subunits. Taking advantage of the unique properties of a commercially available antibody directed against residues 100 -119 within the ␣-helical domain of G␣ s (K-20) and of the peptide corresponding to its target sequence, which are summarized in a recent publication (8), it is shown that the ␤␥-subunits mediating the inhibitory action of isoproterenol were derived from G s .