Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G_s or adenylyl cyclase. Detergent-solubilized G_s from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'-O-(3-thiotriphosphate) (GTPS) when reconstituted into S49 cyc membranes that lack endogenous G_s (carbachol pretreated: GTPS, 93 ± 13% of control; NaF/AlCl₃, 99 ± 8.6% of control; n = 4). Exogenous G_s solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 ± 10% of control; NaF/AlCl₃, 54 ± 11% of control; n = 4). [³H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.