1. We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach. 2. DMPP (1-4 microM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP-induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca2+ dependent, being unaffected at 50 Hz by a 4-fold decrease in the extracellular Ca2+ concentration. 3. The facilitation of evoked ACh release at 50 Hz by 2 microM DMPP was abolished by 10 microM of the calmodulin antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 microM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 microM) to depress evoked ACh release at 50 Hz was also abolished by 10 microM W7. 4. The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin-dependent systems in this facilitatory effect of the compound.