The muscarinic receptor in the rat large airway was characterized by radioligand binding experiments. Using I-quinuclidinyl (phenyl-4-[3H])benzilate ([3H]QNB) as the radioligand, the receptor appears to be homogenous. The receptor density was 23 fmol/mg of protein and the Kd value for [3H]QNB binding was 16 pM. Competition of the [3H]QNB binding for the receptor with selective antagonists and agonists was used to characterize the muscarinic receptor. The K0.5 values for the (M1)-selective antagonists pirenzepine and telenzepine were 210 and 20 nM, respectively. The M2a-selective antagonist AF-DX 116 and the M2b-selective antagonist hexahydrosila-difenidol had K0.5 values of 130 and 120 nM, respectively. By comparing the apparent affinities of these antagonists in the large airways to their affinities in rat heart, the large airway muscarinic receptor appears to be of the M2a type. Agonists competition curves of [3H]QNB binding to the receptor were shallow. The agonist curves were modeled to one- and two-site binding models. All agonists, including M1-selective agonists, gave preferred fits to two-site models. Guanine nucleotide in the assay caused right shifts of the competition curves and decreased the apparent proportion of the receptor population that was in the higher affinity state for the agonists. Thus, it is concluded that: 1) the rat large airway muscarinic receptor interacts with antagonists in a manner which support the hypothesis that the receptors are of the M2a subtype and 2) both the high and low agonist affinity states of the M2a receptor of the rat large airways are capable of interacting with M1 agonists.