The physiological importance of muscarinic M3 and M2 receptors for bladder function was investigated in vivo using mice lacking M3 or M2 receptors and littermate WT controls. Unanesthetized mice of each sex underwent continuous cystometry before and after administration of atropine (1 mg/kg). Male M3 knockout (KO) mice had longer voiding intervals, and larger micturition volumes and bladder capacity than M2 KO or WT males. There was no significant difference in any cystometric parameters between male M2 KO and WT mice. In females M3 KO and M2 KO mice had longer voiding intervals and larger micturition volumes than WT animals. Atropine had marked inhibitory effects on voiding efficacy in WT and M2 KO mice but it had no effect on any cystometric parameters in M3 KO mice. The current results confirm that M3 receptor is the principal muscarinic receptor subtype responsible for bladder contraction and the role of M2 receptors is of minor importance. Functional impairments found in M3 KO mice were milder than those elicited by acute blockade of muscarinic receptors by atropine in WT mice, suggesting that noncholinergic mechanisms can compensate for a chronic loss of M3 receptors.