Release of 3H-ACh from isolated nerve endings of rat hippocampus was evoked by incubation in Krebs-Ringer's buffer containing 25 or 35 mM potassium. The release was Ca2+-dependent and could be inhibited by Mg2+ (20 mM). The muscarinic antagonist, atropine (10(-10)-10(-6) M), enhanced 3H-ACh-release. The muscarinic agonist, carbachol (10(-5)-10(-3) M) inhibited 3H-ACh release via interaction with muscarinic receptors: this effect could be blocked by atropine (10(-6) M). The presence of the feed-back regulation of 3H-ACh release in a cell-free preparation provides further evidence that the presynaptic regulation is exerted by muscarinic autoreceptors localized on the cholinergic nerve ending itself. The feed back inhibition of the 3H-ACh release does not require the presence of intact neurons or neuronal loops as tetrodotoxin (2.5 x 10(-6) M) does not affect the above results.