To elucidate the function of muscarinic receptor subtypes in bladder contractility, we compared the effects of subtype selective muscarinic antagonists on carbachol induced and electric field stimulated contractility of rat bladder in-vitro. The affinity of a series of muscarinic antagonists derived from Schild plot analysis of cumulative carbachol dose response curves indicated M3 mediated bladder contraction. Different affinities were observed with inhibition of electric field stimulated contractions. The M2 selective antagonist methoctramine (10 nM) increased nerve evoked contractions by 17% (p < 0.01) whereas the M1 selective antagonist pirenzepine (PZP, 10 nM) inhibited contractions by 18.5% (p < 0.01). Identical doses had no effect on carbachol induced contractions. Higher doses of all antagonists tested inhibited fully electric field stimulated contractions in a manner consistent with their affinity for M3 receptors. Thse data indicate that presynaptic M1 facilitory and M2 inhibitory receptors and post-synaptic M3 receptors interact to mediate bladder smooth muscle contraction. The presence of m1 receptors in the rat bladder body could not be demonstrated by subtype selective antibodies (only the m2 and m3 subtypes could be detected immunologically) and Northern blots did not reveal the presence of m1 receptor mRNA. However using the reverse transcriptase polymerase chain reaction (RT-PCR) m1, m2, m3, and m4 transcripts were identified in rat bladder. Predicted dose response curves based on a three receptor system were generated. Only the dose response curves generated by incorporating presynaptic M1 facilitory and M2 inhibitory receptors modulating an M3 mediated contractile response resembled the electric field stimulated data. Addition of a presynaptic M4 inhibitory receptor resulted in predicted dose response curves that were nearly indistinguishable from the curves predicted by the three receptor system.