Intracellular recordings from botulinum toxin type A (BoTx)-poisoned extensor digitorum longus muscles from adult rats have shown that the toxin initially reduced the frequency of miniature endplate potentials (m.e.p.ps) to about 1/200 of normal. After a few days the m.e.p.p. frequency rose and was subsequently maintained at a level of about 1/3 of that at normal endplates. Depolarization of the nerve terminals with 20-30 mM KCl-Ringer initially failed to affect the frequency of m.e.p.ps and later caused only a 2-3--fold increase in their frequency. The temperature dependence of m.e.p.p. frequency at BoTx-poisoned endplates had a Q10 of about 12 compared to 2-3 for normal junctions. The time to peak of a population of m.e.p.ps at Botx-poisoned junctions was prolonged as compared to normal and fast- and slow-rising m.e.p.ps originated within the same post-synaptic membrane field area. M.e.p.ps in BoTx-poisoned muscles resembled the m.e.p.ps which 4-aminoquinoline (4-AQ) has been shown to induce in normal muscle, and we therefore examined and compared these two release processes for acetylcholine. Procedures known to markedly affect m.e.p.p. frequency at normal junctions, such as nerve terminal depolarization or changes in extra- and intracellular Ca2+ concentrations, failed to affect m.e.p.p. frequency in BoTx-poisoned muscles and similarly the frequency of m.e.p.ps induced by 4-AQ in normal muscle. Tonicity changes in the extracellular medium altered m.e.p.p. frequency in both the experimental conditions, but in a direction opposite to that at normal junctions. The temperature dependence of the frequency of 4-AQ-induced m.e.p.ps was similar to that of m.e.p.ps at BoTx-poisoned junctions. It is concluded that BoTx poisoning induces an abnormal type of spontaneous quantal transmitter release, characterized by being insensitive to nerve terminal depolarization and to transmembrane Ca2+ fluxes. This transmitter release has characteristics similar to that previously described for the release induced, at normal junctions, by 4-AQ.