1. A quaternary derivative of (-)-hyoscyamine, phenthonium (Phen) induced a concentration-dependent increase in the rate of spontaneous quantal release of acetylcholine (ACh) at the mammalian neuromuscular junction, as shown by intracellular recordings of the miniature endplate potentials (m.e.p.ps) in rat diaphragm muscles. 2. The prejunctional effect of Phen (10-50 microM) was reversible, unrelated to temperature (22 degrees-35 degrees C), unaltered by either changes in [Ca2+]o or by high [Mg2+]o, and was not induced by membrane depolarization. 3. Simultaneously, Phen reduced the amplitude of m.e.p.ps by a postjunctional action. 4. The muscarinic agonist oxotremorine did not prevent the increase in m.e.p.p. frequency induced by Phen. Cholinesterase inhibition with neostigmine potentiated the prejunctional effect induced by a low (20 microM) but not a high (50 microM) concentration of Phen. 5. The increase in m.e.p.p. frequency induced by Phen was not influenced by previous incubation with either atropine (0.01-10 microM) or (+)-tubocurarine (0.05-0.1 microM). Each antagonist however, intensified the postjunctional effect of Phen. 6. Phen (20 microM) did not influence the quantal contents of e.p.ps in cut-muscle preparations or in the presence of high [Mg2+]o. A high concentration of Phen (50 microM) increased the rundown of e.p.p. trains evoked at 10-50 Hz. 7. The results indicate that the facilitatory prejunctional action of Phen cannot be explained by an antimuscarinic activity. A possible interaction of the antagonist with putative prejunctional nicotinic cholinoceptors however, was not excluded.