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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 pD_2 value of 7.1. The concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by the β_1-/β_2-nonselective antagonists propranolol and bupranolol, with pA_2 values of 8.85 and 8.97, respectively. Adrenaline-induced relaxation was not affected by the β_1-selective antagonists atenolol and CGP-20,712A within the concentration ranges supposed to antagonize the β_1-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 pA_2 value of 5.77. The concentration-response curve for adrenaline was also competitively antagonized by the β_2-selective antagonists butoxamine and ICI-118,551 with pA_2 values of 6.86 and 8.73, respectively. The pA_2 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 β_2-selective adrenoceptor agonist. The present findings provide evidence that the relaxant response of the smooth muscle of the guineapig trachea to the adrenal medulla hormone, adrenaline, is mainly mediated through β_2-adrenoceptors.