4-aminopyridine is commonly used to stimulate neurotransmitter release resulting from sustained plasma membrane depolarization and Ca²⁺-influx from the extracellular space. This paper elucidated unconventional mechanism of 4-aminopyridine-stimulated glutamate release from neurons and non-neuronal cells which proceeds in the absence of external Ca²⁺. In brain nerve terminals, primary neurons and platelets 4-aminopyridine induced the exocytotic release of glutamate that was independent of external Ca²⁺ and was triggered by the sequestration of Ca²⁺ from intracellular stores. The initial level of 4-aminopyridine-stimulated glutamate release from neurons in the absence or presence of external Ca²⁺ was subequal and the difference was predominantly associated with subsequent tonic release of glutamate in Ca²⁺-supplemented medium. The increase in [Ca²⁺]_i and the secretion of glutamate stimulated by 4-aminopyridine in Ca²⁺-free conditions have resulted from Ca²⁺ efflux from endoplasmic reticulum and were abolished by intracellular free Ca²⁺ chelator BAPTA. This suggests that Ca²⁺ sequestration plays a profound role in the 4-aminopyridine-mediated stimulation of excitable and non-excitable cells. 4-Aminopyridine combines the properties of depolarizing agent with the ability to sequester intracellular Ca²⁺. The study unmasks additional mechanism of action of 4-aminopyridine, an active substance of drugs for treatment of multiple sclerosis and conditions related to reduced Ca²⁺ efflux from intracellular stores.