We have studied acetylcholine (ACh) desensitization of Cl- secretion and its reactivation by atropine in the hen tracheal epithelium employing voltage-clamp technique. Adding 4 microM ACh to the serosal side induces a maximal bumetanide-sensitive Cl- secretion. The ACh-induced Cl- secretion is desensitized nearly 100% at 512 microM ACh. The desensitization of Cl- secretion may be reduced 30-40% by 0.32 microM atropine (reactivation). From a kinetic model for desensitization and reactivation with two muscarinic receptors (mAChR), we have estimated the maximal secretory response, the apparent dissociation coefficients, and the apparent Hill coefficients for ACh and atropine at the two receptors. Based on the kinetic model we can predict an optimal fixed concentration ratio (Ofcor) between ACh and atropine that will maintain Cl- secretion above 40% of maximum over a wide concentration range, ACh 1-4,100 microM, and we have confirmed the predicted Ofcor experimentally. A cellular model is proposed with two mAChR, one stimulatory and another inhibitory, that accounts for an observed dissociation between desensitization in short-circuit current and conductance.