The influence of astrocyte swelling on the cytosolic free calcium concentration [Ca 21 ] i was studied at the single cell level. Sudden exposure of normo-osmotically (305 mosmol/l) cultured astrocytes to hypo-osmotic medium induced a biphasic increase in cytosolic calcium with an initial peak followed by a sustained plateau. The response was osmolarity dependent and was maximal at 205 mosmol/l with respect to [Ca 21 ] i and the percentage of responding cells. Other modes of astrocyte swelling [gradual adjustment of hypo-osmolarity, normo-osmotic exposure of hyper-osmotic (405 mosmol/l) maintained cells] produced a much weaker [Ca 21 ] i response. Change from 405 to 205 mosmol/l, however, resulted in the entire peak and an increased plateau. Experiments with Ca 21 -free medium and after pretreatment with BAPTA-AM, thapsigargin, phorbol myristate acetate, or nimodipine revealed that the peak mainly resulted from depletion of intracellular Ca 21 stores, whereas the plateau was probably due to capacitative Ca 21 entry and Ca 21 influx independent of store depletion including a nimodipin-sensitive component. Prior depletion of ryanodine-, bradykinin-or ATP-sensitive stores revealed that the initial hypo-osmolarity-induced Ca 21 -release was from a Ca 21 pool also affected by ATP and bradykinin, but not by ryanodine. The recent finding, that the hypo-osmolarityinduced [Ca 21 ] i response was completely maintained if phospholipase C-mediated phosphatidylinositol hydrolysis was blocked, suggests that hypo-osmolarity may exert an inositol (1,4,5) triphosphate-independent access to these stores.