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<em><strong>Objective(s):</strong></em> Baicalein, a compound extracted from a variety of herbs, showed various pharmacological effects. This study evaluated the relaxant effects of baicalein and its underlying molecular mechanisms of action on rat’s isolated tracheal smooth muscle.<br /><em><strong>Materials and Methods:</strong></em> Tracheal smooth muscle were contracted by 10 μM methacholine or 60 mM KCl and the effects of cumulative concentrations of baicalein (5, 10, 20 and 40 mg/ml) and theophylline (0.2, 0.4, 0.6 and 0.8 mM) were evaluated. To examine the possible mechanism(s) of the relaxant effect of baicalein, its effect was also evaluated on incubated tissues with atropine, indomethacin, diltiazem, N(G)-Nitro-L-arginine methyl ester (L-NAME), glibenclamide, propranolol and chlorpheniramine.<br /><em><strong>Results:</strong></em> A concentration-dependent and significant relaxant effect was seen for baicalein in non-incubated tissues contracted by KCl or methacholine (P<em><strong>Conclusion:</strong></em> A potent relaxant effect comparable to the effect of theophylline was shown for baicalein, which was probably mediated via inhibition of histamine (H1) receptors, stimulation of beta2-adrenergic receptors and potassium channels activation.