M3muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2receptors participate in contraction because M3-selective antagonists [ para-fluoro-hexahydro-sila-diphenidol ( p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2receptor and one by the M3receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine: p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2and M3receptors can induce contraction. In the denervated bladder, the M2and the M3receptors interact in a facilitatory manner to mediate contraction.