1. The effects of phenthonium and related compounds on the spontaneous release of acetylcholine (ACh) were investigated with electrophysiological and radiolabelled techniques to correlate the prejunctional effect with their cholinolytic activities and to determine the structure-activity relationship. 2. Phenthonium and endophen are N-(4-phenyl)-phenacyl derivatives of l-hyoscyamine in "exo" and "endo" conformation, respectively. Tropol is N-(4-phenyl) phenacyl tropan-3-ol whereas ipratropium is 8-isopropyl-noratropine. 3. Only phenthonium increased the frequency of miniature endplate potentials and the resting efflux of spontaneous [3H]-ACh in rat diaphragm muscles. 4. The rank order of the antimuscarinic potency was: ipratropium > atropine > phenthonium = endophen > tropol. The rank order of the antinicotinic activity was: phenthonium = endophen > tropol > atropine > ipratropium. 5. It is concluded that the prejunctional facilitatory effect of phenthonium is associated with the N-phenyl-phenacyl group at "exo" conformation but the effect is unrelated to its cholinolytic properties.