The interaction between theophylline (T) and the beta-agonists albuterol (A) and isoproterenol (I) was examined using canine cervical tracheal smooth muscle devoid of epithelium contracted with 0.1 or 0.3 microM methacholine. Greater functional antagonism with beta-agonists vs. T was confirmed and an ability of T to potentiate beta-agonist relaxation was demonstrated. The EC50 for T increased from 0.13 +/- 0.02 to 0.37 +/- 0.07 mM (mean +/- SEM) in preparations contracted with 0.1 or 0.3 microM methacholine, respectively, while that for I increased from 0.036 +/- 0.008 to 0.17 +/- 0.03 microM, a significantly larger change (P < 0.025). In tissues contracted with 0.3 microM methacholine and pretreated with 10 micrograms/ml of T IC50 values from composite concentration-response curves for I and A were displaced to the left and Emax was increased (56.6 to 71.5% for I, 44 to 61% for A, P < 0.0002). Addition of 10 micrograms/ml T resulted in relaxations which exceeded that calculated by the fractional product method for additive, independent action (P < 0.0001 for I, P < 0.0002 for A at 0.3 microM methacholine), suggesting that at least part of T's action was over-additive. Five, 10 and 20 micrograms/ml T enhanced the effectiveness of single concentrations of I by factors of 1.47 +/- 0.14 (P < 0.05), 2.72 +/- 0.26 (P < 0.01) and 5.34 +/- 0.55 (P < 0.01), respectively, in preparations contracted with 0.1 microM methacholine: I enhanced the effectiveness to a lesser degree. Using two approaches, positive interaction or over-additivity between T and beta-agonists has been demonstrated.