In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats that had their major pelvic ganglion bilaterally removed. Denervation induced both hypertrophy and a supersensitivity of the bladders to agonist. The affinities in control bladders for antagonism of carbachol-induced contractions were consistent with M3-mediated contractions. Affinities in denervated bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (8.5) and p-fluoro hexahydrosilodifenidol (6.6) were consistent with M2-mediated contractions, although the methoctramine affinity (6.5) was consistent with M3-mediated contractions. Subtype-selective immunoprecipitation of muscarinic receptors revealed a 50% increase in total and a 60% increase in M2 receptor density with no change in M3 receptor density in denervated bladders compared with normal or sham-operated controls. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol-induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediates smooth muscle contraction in the denervated bladder.