We studied the effect of pertussis toxin (PT) and partial muscarinic antagonism using pirenzepine (PIR) on beta-adrenergic relaxation of muscarinic contraction in 188 tracheal smooth muscle (TSM) preparations from 25 dogs in vitro. Strips of TSM were incubated for 4 h at 37 degrees C in Krebs-Henseleit (K-H) perfusate with or without 10 micrograms/ml of PT. In tissues contracted to target tension (TT; 50% of maximal response to 127 mM potassium-substituted K-H [KCl]) with acetylcholine (ACh), pretreatment with PT decreased the concentration of isoproterenol (ISO) causing 30% relaxation from TT (RC30) from 1.3 +/- 0.8 x 10(-7) M (control) to 2.8 +/- 0.7 x 10(-8) M (p = 0.013). Pretreatment with PT also augmented the maximal relaxation elicited by 10(-5) M ISO. In separate studies, strips of TSM were contracted with ACh; pretreatment with 10(-7) M PIR decreased the concentration of ISO causing 50% relaxation (RC50) from 3.4 +/- 0.6 x 10(-7) to 9.6 +/- 1.5 x 10(-8) M (p = 0.042). Pretreatment with PIR did not affect relaxation elicited by ISO for strips contracted equivalently with KCl. In addition, PIR increased both the potency and efficacy of ISO in relaxing muscarinic contraction in sham-incubated strips of TSM but had no effect after incubation with PT. Neither PT nor PIR affected beta-adrenergic relaxation of TSM contracted with KCl. Our data demonstrate that beta-adrenergic receptor relaxation of muscarinic contraction is augmented by (1) incubation with PT and (2) partial blockade of muscarinic receptors.