Cadmium ion (Cd2+) was examined for effects on frequency of miniature end-plate potentials (m.e.p.p.s) under several conditions in mouse diaphragm muscles. Cd2+ depressed potassium (K+)-stimulated rise in m.e.p.p. frequency, acting antagonistically to Ca2+, whereas in the absence of K+ stimulation Cd2+ produced a time- and concentration-dependent rise in m.e.p.p. frequency without affecting the resting membrane potential of muscle fibers. Such a presynaptic effect was observed with or without external Ca2+ [( Ca]0). In the absence of [Ca]0, Cd2+ frequently produced twitching in several fibers, which caused dislodgement of the micro-electrode. Therefore, 10 mM KCl was added to the Ca-free solution to overcome such difficulty. The following experiments were performed in the Ca-free 10 mM KCl solution. Prolonged exposure to Cd2+ led to an eventual decline in m.e.p.p. frequency within 20-30 min. After decline of the effect, m.e.p.p. frequency in the presence of Cd2+ was not restored by adding Ca2+ combined with elevated K+ and with A23187 or by carbonyl cyanide p-trifluoromethoxyphenyl hydrazine and hypoxic condition, each of which was separately effective in enhancing transmitter release. Exposure to 50 microM Cd2+ for 60 min decreased acetylcholine content in diaphragm muscle to approximately 50% of control. Tetrodotoxin and caffeine did not modify the presynaptic stimulatory effect of Cd2+. The effect of Cd2+ on m.e.p.p. frequency was partially reduced by trifluoperazine and phenytoin each of which depressed m.e.p.p. frequency rise induced by Ca2+ with elevated K+. The possible mechanism of Cd2+ action and of its interaction with intracellular Ca2+ stores is discussed.