In mammalian nerve-muscle preparations treated with an anticholinesterase, the acetylcholine (ACh) released non-quantally (NQR) reaches the postsynaptic receptors and causes a small depolarization of the membrane potential at the endplate region of the muscle fibres. Increase in quantal release potentiates the NQR and vice versa, the amplitude and the kinetic parameters of quantal miniature endplate currents (MEPCs) change during manipulation of NQR, indicating direct interaction between both types of release. Repetitive binding of ACh to postsynaptic receptors which prolongs the time course of MEPCs in anti-cholinesterase-treated endplates leads within 1-2 h to progressive desensitization in the presence of non-quantal release and to the subsequent shortening of the quantal responses. We have also investigated the effect of procedures known to modulate non-quantal acetylcholine release, on the small, but obvious, difference in the resting membrane potential between the endplate zone and other areas of the mouse muscle fibre. The resting membrane potential at the endplate zone with intact cholinesterase is more negative (by 2-4 mV) than in the endplate-free area. The experiments were performed to test the hypothesis that the hyperpolarization is caused by an electrogenic Na(+)-K+ pump operating during the action of ACh released in non-quantal form. Observations in favour of this idea are that both short-term denervation (which eliminates non-quantal but not quantal release) and ouabain abolish the local synaptic hyperpolarization and that subsequent application of low doses of ACh restores it. It follows, therefore, that the hyperpolarization is probably caused by a small but continuous ACh leakage from the nerve terminal.