Aims To investigate the role of the mucosa in (-)-isoprenaline-induced relaxation of mouse detrusor muscle and to characterize the β-adrenoceptor subtypes involved. Methods Isolated intact and mucosa-denuded muscle strips from the urinary bladder of male C57BL6 mice were pre-contracted with KCl (40mM) and were relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin in the presence and absence of the subtype-selective β-AR blockers CGP20712A (β₁-ARs), ICI118,551 (β₂-ARs), and L748,337 (β₃-ARs). Results Force development in response to KCl was larger in mucosa-denuded than in intact preparations and was almost completely relaxed with increasing concentrations of (-)-isoprenaline. Mucosa-denuded muscles were about 10-fold more sensitive to (-)-isoprenaline than intact muscles. CGP20712A did not affect the concentration-response curves (CRCs) to (-)-isoprenaline, ICI118,551 shifted the CRC further to the right in denuded than in intact strips so that the difference between them was abolished. Combined exposure to β₁-AR and β₂-AR blocker yielded the same result. L748,337 did not significantly affect the CRC to (-)-isoprenaline but caused additional blockade to ICI118,551 in the presence of intact mucosa. Conclusions The mucosa of mouse detrusor strips impairs KCl-induced force development and reduces the sensitivity to β-AR-induced relaxation. The relaxing response to (-)-isoprenaline as well as the mucosa effect thereupon are mainly mediated by β₂-ARs. A minor involvement of β₃-ARs becomes apparent particularly at high (-)-isoprenaline concentrations.