The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [3H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [3H]QNB. Specific binding was time- and temperature-dependent. The association of [3H]QNB with the muscarinic receptor reached equilibrium much sooner at 37 degrees C than 25 degrees C at a [3H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [3H]QNB concentration of 1800 pM. All remaining experiments were performed at 37 degrees C. Binding was saturable with respect to [3H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 51 +/- 20 pM and a maximum receptor density (Bmax) of 2.17 +/- 0.27 pmole/mg protein. The Hill coefficient for [3H]QNB binding was 1.07 +/- 0.16. The association (K1) and dissociation (K-1) rate constants were determined to be (5.51 +/- 0.16) X 10(8) M-1 min-1 and (1.41 +/- 0.18) X 10(-2) min-1, respectively. KD calculated from the ratio of K1 and K-1 was 26.3 +/- 3.8 pM; this value is close to the value of KD calculated from Scatchard plots of binding isotherms. The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20 +/- 0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle. The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [3H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (Ki) of nanomolar range. Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave Ki values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [3H]QNB binding sites of the M2 subtype for all tested muscarinic antagonists. Competition with selective muscarinic agonists pilocarpine and carbachol yielded Ki values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P less than 0.01).