In the guinea-pig vas deferens, the contractions induced by pilocarpine and arecoline, unlike ACh, were markedly reduced by treatment with procaine (10(-6) g/ml) or hemicholinium (5 x 10(-4) g/ml). These reductions by hemicholinium were completely recovered after exposure to choline. The contractile responses to these three agonists were eliminated by atropine (3 x 10(-8) g/ml) but enhanced by neostigmine (3 x 10(-8) g/ml), though these were not affected at all by tetrodotoxin (3 x 10(-8) g/ml) or hexamethonium (10(-5) g/ml). The contractile responses to ACh, pilocarpine and arecoline were potentiated by ouabain (10(-6) g/ml). These potentiations were more marked in pilocarpine and arecoline than those in ACh. The potentiation in response to arecoline was maximal after incubation with ouabain for 5 min but that to ACh for 45 min. The results suggest that pilocarpine and arecoline act as ACh-releaser in this tissue and that the ouabain-induced cholinergic potentiation may be attributable to a dual mechanism, facilitation of ACh release at presynaptic site and alteration of the membrane excitability at postsynaptic site.