Background and Purpose Mirabegron is the first β3-adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β3-adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α1-adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α1A- (vas deferens and prostate), α1D- (aorta) and α1B-adrenoceptors (spleen). Experimental Approach Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [3H]prazosin to membrane preparations of HEK-293 cells expressing each of the human α1-adrenoceptors, as well as β-adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed. Key Results Mirabegron produced concentration-dependent urethral relaxations that were shifted to the right by the selective β3-adrenoceptor antagonist L-748,337 but unaffected by β1- and β2-adrenoceptor antagonists (atenolol and ICI-118,551 respectively). Mirabegron-induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α1-adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α1-adrenoceptors in urethra, vas deferens and prostate (α1A-adrenoceptor, pA2 ≃ 5.6) and aorta (α1D-adrenoceptor, pA2 ≃ 5.4) but not in spleen (α1B-adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α1A- and α1D-adrenoceptors (pKi ≃ 6.0). 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