Objectives: This study examines whether M2 receptors contribute to direct contraction of the detrusor in human neurogenic and idiopathic overactive bladders. Methods: Control detrusor muscle was obtained from patients undergoing cystectomy for bladder cancer, whilst overactive detrusor muscle was obtained from patients undergoing clam cystoplasty for idiopathic or neurogenic detrusor overactivity. The affinities of a range of subtype selective antagonists (DAMP, darifenacin, methoctramine R0-320-6206, and pirenzepine) were obtained in tissue bath experiments by using carbachol as the agonist. These affinity values were then compared with the known affinities for these antagonists at the muscarinic receptor subtypes. Results: An increased sensitivity to carbachol was observed in both the neurogenic and idiopathic overactive detrusors compared with the control human detrusor. The M2-selective antagonists (methoctramine, R0-320-6206) and M1-selective antagonist (pirenzepine) had low affinities, whilst the M3-selective antagonists (4-DAMP and darifenacin) had high affinities for the human detrusor muscarinic receptor in all three groups of tissues. The affinities (pKB values) for the five antagonists were consistent with antagonisms at the M3 receptor in all three groups; Schild plot analysis indicated an action at this single receptor subtype. Conclusions: Contraction mediated by muscarinic receptors is enhanced in idiopathic and neurogenic overactive detrusors compared with control detrusor. The direct contractile response to carbachol is mediated by the M3 receptor in both human normal and overactive bladders, indicating no change in receptor subtype contribution to contraction in the disease state.