Longitudinal muscle strips of the guinea-pig ileum were incubated with [3H]choline and the effects of muscarinic agonists on smooth muscle contraction and on spontaneous and electrically-evoked outflow of tritium were studied. Muscarine and pilocarpine concentration-dependently increased both muscle contraction and spontaneous outflow of [3H]ACh, and inhibited the electrically-evoked outflow of [3H]ACh. The increase in spontaneous outflow was prevented by tetrodotoxin and scopolamine, but not by hexamethonium. Oxotremorine (1-100 microM) did not increase the spontaneous outflow of tritium. Pirenzepine in concentrations of 10 and 100 nM hardly affected the muscle contractions induced by pilocarpine, but significantly antagonized the pilocarpine-evoked increases in [3H]ACh outflow. Likewise, pirenzepine (100 nM) antagonized more effectively the enhancement by muscarine of spontaneous outflow than the inhibitory effect of muscarine on the electrically-evoked release of [3H]ACh. Scopolamine (1 and 10 nM) antagonized to a similar extent the effects of pilocarpine on spontaneous outflow of [3H]ACh and on muscle contraction. The results suggest that the cholinergic nerves of the myenteric plexus are endowed with excitatory (ganglionic) and inhibitory (prejunctional) muscarine receptors which modulate the release of ACh and which differ in their affinities to pirenzepine.