Beta adrenoceptor subtypes in canine tracheal smooth muscle have been investigated by radioligand binding and by physiological responses to beta agonists and sympathetic nerve stimulation in vitro. Specific binding of [3H]dihydroalprenolol to tracheal smooth muscle membranes was of high affinity (Kd = 1.0 +/- 0.08 nM), as in peripheral lung membranes from the same animals, but the concentration of binding sites (95.6 +/- 4.7 fmol/mg of protein) was much lower than in lung (532 +/- 48 fmol/mg of protein). Binding was stereoselective and agonists competed with the rank order of potency isoproterenol greater than epinephrine greater than norepinephrine, signifying a preponderance of beta-2 receptors. Using selective beta antagonists, we determined the ratio of beta-1/beta-2 receptors in tracheal smooth muscle membranes to be 1:4. The relaxation response of tracheal smooth muscle strips to exogenous beta agonists was mediated by beta-2 receptors, with a very small contribution from beta-1 receptors. However, the relaxation response to electrical field stimulation of sympathetic nerves was mediated predominantly by beta-1 receptors. Our results suggest that most beta receptors in dog tracheal smooth muscle are of the beta-2 subtype and mediate responses to circulating catecholamines, but there is a small concentration of beta-1 receptors which mediate the response to neurally released norepinephrine.