The mean miniature end-plate potential (m.e.p.p.) frequency and the acetylcholinesterase (AChE) activity have been examined in the rat diaphragm, in relation to the time of Li⁺ action. Substitution of LiCl for NaCl caused increase in m.e.p.p. frequency, the final level of which depended on Li⁺ concentration in the bath solution. Incubation of diaphragm homogenate with NaCl caused initially a slight increase of AChE activity, reaching a plateau after 20min. Substitution of LiCl for NaCl produced an increase in AChE activity to a maximum, followed by a decrease to a minimum. The activity consequently returned to a steady level. During complete substitution of LiCl for NaCl, the maximum value of AChE activity coincided with the maximum m.e.p.p. frequency (F_max) induced by Li⁺; the minimum value correlated with the precise instant that the frequency reached a plateau. The addition of prostigmine caused: a) blockade of AChE activity, and b) prostigmine concentration-dependent decrease of F_max and/or disappearance of m.e.p.p.s from neuromuscular junctions treated with Li⁺. These results are discussed in terms of correlation between changes of AChE activity induced by Li⁺ and variations of spontaneous quantal release of the transmitter from nerve terminals of the rat diaphragm treated with Li⁺.