Brief transmural stimuli, which selectively excited cholinergic fibres, initiated contractions and excitatory junction potentials in preparations of longitudinal muscle isolated from the guinea-pig ileum: these responses were associated with an increase in the internal concentration of calcium ions. When muscle voltage-dependent calcium channels were blocked using the organic calcium antagonist nifedipine, brief stimuli continued to initiate contractions, evoke excitatory junction potentials and cause an increase in the intracellular calcium concentration. Ionophoretically applied acetylcholine caused depolarizations which resembled the excitatory junction potentials evoked by cholinergic nerve stimulation. Both responses had slow time courses and were abolished by muscarinic receptor antagonists. However, the depolarizations produced by ionophoretically applied acetylcholine, unlike those produced by nerve stimulation, were frequently interrupted by transient hyperpolarizations. The transient hyperpolarizations were abolished by barium ions or charybdotoxin. High concentrations of the calcium antagonists nicardipine, verapamil or diltiazem had a tendency to preferentially abolish the excitatory junction potential. When the effects of the cholinesterase inhibitor, eserine, on excitatory junction potentials were examined, it became apparent that when the destruction of acetylcholine was prevented it initiated an additional conductance change to that initiated by acetylcholine in untreated tissues. The results are discussed in relation to the idea that neuronally released acetylcholine and applied acetylcholine might activate different subsets of muscarinic receptors on longitudinal ileal smooth muscle.