The interaction of β₁- and β₃-adrenergic receptors and G_i proteins was examined in rat adipocytes. In intact adipocytes, cyclic AMP accumulation stimulated by the β₃-selective agonist, BRL 37344 (BRL), was potentiated by pertussis toxin (PTX), as was the β₁-sensitive component of isoproterenol (ISO)-stimulated cyclic AMP accumulation. These data suggest that β₁- and β₃-receptors interact with both G_s and G_i in intact adipocytes. F analysis of the activation of adenylyl cyclase by the β-receptor subtypes was performed in adipocyte membranes in which the activity of G_i was manipulated by both GTP and PTX. Unlike cyclic AMP accumulation in cells, the activation of membrane adenylyl cyclase by ISO could be clearly resolved into components mediated by β₁-(high affinity) to 0.1 μM GTP, but the activity mediated by β₁-receptors was not. As a consequence, the proportion of total ISO-stimulated activity that was mediated by β₃-receptors was significantly reduced at concentrations of GTP in which G_i proteins are active. Adenylyl cyclase activity stimulated by BRL was also inhibited at high concentrations of GTP. PTX abolished the inhibition of β₃-receptor-stimulated activity by high GTP concentrations. This is the first study to indicate that G_i proteins can limit β₃- but not β₁-stimulated adenylyl cyclase activity and are consistent the hypothesis that β₃-receptors interact with both G_s and G_i, whereas β₁-receptors couple predominantly to