Combined patch clamp and Ca2+-measurements (Fura-2) were used to study the dependence of volume-activated Cl--currents (ICl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca2+-concentration [Ca2+]i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 microM BAPTA-AM caused a substantial decrease of ICl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca2+]i was lowered at the peak of the volume-activated current, but this low Ca2+-concentration also prevented activation of the current by a second hypotonic challenge.ICl,vol is already maximally activated at intracellular Ca2+-concentrations between 50 and 100 nmol/l, a further increase of [Ca2+]i does not affect the size of ICl,vol.These results indicate that a sustained full activation of ICl,vol in endothelial cells requires submicromolar concentrations of Ca2+, and that changes in [Ca2+]i do not modulate the current.