Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca2+ signalling, but the impact of transient Ca2+ signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca2+ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of −100 mV, voltage steps to −180 mV triggered elevation of the cytosolic free Ca2+ concentration. The increase in the cytosolic Ca2+ level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca2+ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of −349 pA (SE = 107) at −100 mV. Ca2+ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to −100 mV, a transient increase in the cytosolic Ca2+ level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca2+ elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca2+ transport proteins, resulting in an overshoot of the cytosolic Ca2+ level after returning the membrane potential to −100 mV.