1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.