In airway smooth muscle, muscarinic agonists inhibit synthesis of adenosine 3',5'-cyclic monophosphate (cAMP). The goal was to characterize the relationship between agonist occupancy of muscarinic receptors and regulation of cAMP for bovine trachealis cells. For intact cells dispersed by enzyme, carbachol maximally inhibited 58 +/- 4% (mean +/- SE, n = 5) of isoproterenol-stimulated cAMP accumulation at low concentrations [log half-maximal effective concentration (EC50) = -8.34 +/- 0.16]. In radioligand binding experiments, carbachol competed for [3H]quinuclidinyl benzilate (n = 7) and [N-methyl-3H]scopolamine (n = 3) binding sites on intact cells with both low (log KL = -4.26 +/- 0.06 and -4.50 +/- 0.20, respectively) and high affinities (log KH = -5.91 +/- 0.24 and -6.39 +/- 0.19, respectively). In separate experiments, a fraction of the muscarinic receptors on intact cells were inactivated with either phenoxybenzamine (POB) or propylbenzylcholine mustard (PBCM). We compared equally effective concentrations of carbachol before and after partial inactivation of receptors, and the calculated equilibrium dissociation constants for agonist (log KA = -4.36 +/- 0.42 to -3.20 +/- 0.40 for POB; log KA = -4.27 +/- 0.45 for PBCM) were much greater than the half-maximally effective concentration of carbachol in control cells (log EC50 = -8.34 +/- 0.16). Based on these equilibrium dissociation constants, we calculated that maximum responses (EC95) to carbachol were obtained by occupancy of 0.8% of the receptors coupled to cAMP regulation. We concluded that muscarinic inhibition of cAMP accumulation is characterized by a muscarinic receptor reserve.