The β-adrenoceptor subtype that mediates adrenaline-induced relaxation was pharmacologically identified in smooth muscle cells of the isolated guinea-pig trachea. Adrenaline produced a concentration-dependent relaxation with a pD₂ value of 7.1. The concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by the β₁-/β₂-nonselective antagonists propranolol and bupranolol, with pA₂ values of 8.85 and 8.97, respectively. Adrenaline-induced relaxation was not affected by the β₁-selective antagonists atenolol and CGP-20,712A within the concentration ranges supposed to antagonize the β₁-subtype (atenolol, ≤ 10^-6 M; CGP-20,712A, ≤ 10 ^-8 M). By contrast, the concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by atenolol at concentrations ≥ 3 × 10^-6 M with a pA₂ value of 5.77. The concentration-response curve for adrenaline was also competitively antagonized by the β₂-selective antagonists butoxamine and ICI-118,551 with pA₂ values of 6.86 and 8.73, respectively. The pA₂ values of β-adrenoceptor antagonists (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551) tested against adrenaline were consistent with the values when tested against salbutamol, a β₂-selective adrenoceptor agonist. The present findings provide evidence that the relaxant response of the smooth muscle of the guinea-pig trachea to the adrenal medulla hormone, adrenaline, is mainly mediated through β₂-adrenoceptors.