β-ARs are sub-classified into three subtypes (β₁–β₃). Among these, β₃-ARs are present in various types of SM including DSM and are believed to play a role in relaxations of these muscles. To date, there has been little information available about the endogenous ligand that stimulates β₃-ARs to produce relaxations in DSM. In this study, to determine whether NA is an endogenous ligand of DSM β₃-ARs, NA-induced relaxation was pharmacologically analysed using rat DSM. In isolated rat urinary bladder tissues, mRNAs for β₁-, β₂-, and β₃-ARs were detected using RT-PCR. In DSM preparations contracted with methacholine (3 × 10⁻⁵ M), NA-induced relaxation was not inhibited by atenolol (10⁻⁶ M), ICI-118,551 (3 × 10⁻⁸ M), propranolol (10⁻⁷ M), and bupranolol (10⁻⁷ M). In the presence of propranolol (10⁻⁶ M), NA-induced relaxation was competitively inhibited by bupranolol (3 × 10⁻⁷–3 × 10⁻⁶ M) or SR59230A (10⁻⁷–10⁻⁶ M), with their pA₂ values being 6.64 and 7.27, respectively. None of the six NA metabolites showed significant relaxation in methacholine-contracted DSM. These findings suggest that NA, but not its metabolites, is an endogenous ligand for β₃-ARs to produce relaxations of DSM in rats.