1 Atropine (10(-5) M) enhanced the release of [3H]-acetylcholine from rat isolated hemidiaphragms, previously incubated with [3H-methyl]-choline, stimulated via their phrenic nerves. 2 Oxotremorine (10(-5) M) did not affect the stimulated release of [3H]-acetylcholine but antagonized the facilitatory effects of atropine (10(-5) M). 3 It is suggested that there are presynaptic inhibitory muscarinic receptors that modulate the release of acetylcholine in the phrenic nerves of the rat.