The relationship between 86Rb+ distribution across synaptosomal membrane and [14C]acetylcholine (ACh) release have been studied in a rat brain cortex synaptosomal preparation using K+, ouabain and veratridine depolarization. Decrease in membrane potential, approximated from the 86Rb+ distribution, is accompanied by an increase in [14C]ACh release, but the extent of the increase at a certain depolarization is dependent on how the depolarization is induced. A substantial depolarization by K+ is necessary to enhance ACh release, as compared to ouabain and veratridine where only a slight depolarization is accompanied by an increase in ACh release. In Ca2+-free, EGTA-containing medium ouabain and veratridine can also increase [14C]ACh release. The relationship between membrane potential and ACh release is very similar in the presence of ouabain and veratridine both in Ca2+-containing and Ca2+-free medium. The effect of ouabain and veratridine on the Na-K exchange pump is different; ouabain can completely abolish Na-K-ATPase activity and 86Rb+ uptake of synaptosomes, whereas veratridine does not seem to influence the activity of the pump. m-Chloro-carbonylcianid phenyl hydrazon (50-500 nM) increases [14C]ACh release in a concentration-dependent manner without a considerable change of membrane potential or Na-K pump activity. The Ca2+ ionophore A 23187 induces a substantial increase in [14C]ACh release in the absence of external Ca2+. In this case neither Na-K pump activity nor membrane potential of synaptosomes is changed. A possible role of intracellular Ca2+ mobilization as a consequence of increased intracellular Na+ concentration in some depolarization-induced transmitter release is discussed.