The inhibition of preganglionic and postganglionic contractions of the rabbit isolated bronchus/trachea by antagonists with selectivity for different muscarinic receptor subtypes was compared with their affinities at M1, M2, M3 and M4 receptors. Neither M1/M3 receptor-unselective antagonists (atropine, hexahydro-siladifenidol, thiazinamium, p-fluoro-hexahydro-sila-difenidol) nor antagonists with selectivity for the M1 over M3 subtype ((+)-biperiden, UH-AH 37, telenzepine, o-methoxy-sila-hexocyclium, pirenzepine) consistently showed a preferential inhibition of the response to preganglionic over postganglionic stimulation. Potencies for inhibition of contraction to preganglionic stimulation by antagonists discriminating more than threefold both between M1 and M3, and between M3 and M2 receptors (hexocyclium, (+)-biperiden, UH-AH 37, telenzepine, o-methoxy-silahexocyclium, p-fluoro-hexahydro-sila-difenidol, pirenzepine) are most consistent with affinities at smooth muscle M3 receptors as determined on methacholine-contracted rabbit trachea. Antagonists with a 10-fold higher affinity at M2 over M3 receptors enhanced contractions to field stimulation (AQ-RA 741 = AF-DX 384 = idaverine > himbacine = imperialine = AF-DX 116 = methoctramine >> gallamine), whereas antagonists with a selectivity profile of M4 > or = M3 > M2 (hexahydro-sila-difenidol, pirenzepine, dicyclomine) failed to increase the contractions. Secoverine (selectivity profile M4 > M2 > M3) enhanced contractions at concentrations consistent with its M2 receptor affinity. These results (1) exclude a ganglionic M1 receptor modulating excitatory vagal neurotransmission but (2) suggest the presence of an inhibitory prejunctional M2 rather than an M4 receptor and (3) identify a postjunctional smooth muscle M3 receptor in rabbit bronchus/trachea.