We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 μmol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 μmol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 μmol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 μmol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.