β-Adrenoceptors are subclassified into 3 subtypes (β₁–β₃). Among these, β₃-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. β₃-Adrenoceptors are also present in urinary bladder smooth muscle (UBSM), although their expression varies depending on the animal species. To date, there has been little information available about the endogenous ligand that stimulates β₃-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM β₃-adrenoceptors, noradrenaline-induced relaxation was analyzed pharmacologically using rat UBSM. We also assessed whether noradrenaline metabolites were ligands in UBSM. In isolated rat urinary bladder tissues, mRNAs for β₁-, β₂-, and β₃-adrenoceptors were detected using RT-PCR. In UBSM preparations contracted with methacholine (3 × 10⁻⁵ M), noradrenaline-induced relaxation was not inhibited by the following antagonists: atenolol (10⁻⁶ M; selective β₁-adrenoceptor antagonist), ICI-118,551 (3 × 10⁻⁸ M; selective β₂-adrenoceptor antagonist), propranolol (10⁻⁷ M; non-selective β-adrenoceptor antagonist), and bupranolol (10⁻⁷ M; non-selective β-adrenoceptor antagonist). In the presence of propranolol (10⁻⁶ M), noradrenaline-induced relaxation was competitively inhibited by bupranolol (3 × 10⁻⁷–3 × 10⁻⁶ M) or SR59230A (10⁻⁷–10⁻⁶ M; selective β₃-adrenoceptor antagonist), with their p A ₂ values calculated to be 6.64 and 7.27, respectively. None of the six noradrenaline metabolites produced significant relaxation of methacholine-contracted UBSM. These findings suggest that noradrenaline, but not its metabolites, is a ligand for β₃-adrenoceptors to produce relaxation responses of UBSM in rats.