In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in 1-2 week old Sprague - Dawley rat bladders were not affected by M₂ or M₃ antagonists. However administration of physostigmine (0.5 µM), an anticholinesterase agent that increases the levels of endogenous acetylcholine, or carbachol (50-100 nM), a cholinergic agonist at low concentrations which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M₂ receptors with AF-DX 116 (0.1 µM) or methoctramine (1µM), or blockade of M₃ receptors with 4-DAMP (50 nM) or 4-DAMP mustard (0.1 µM) reversed the physostigmine and carbachol responses. M₂ and M₃ blockade did not alter the facilitation of spontaneous contractions induced by Bay K-8644 (10 nM), an L-type calcium channel opener, or iberiotoxin (0.1 µM), a BK_Ca channel blocker. NS1619 (30 µM), a BK_Ca channel opener, decreased carbachol augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of both M₂ and M₃ receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.