The calcium (Ca 2ุ ) dependence of potassium (K ุ ) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments 86 Rb release and changes in cytosolic Ca 2ุ ([Ca 2ุ ] i ). Hyposmotic (50%) medium increased [Ca 2ุ ] i from 117 to 386 nM, with contributions of extracellular Ca 2ุ and Ca 2ุ from the endoplasmic reticulum. Hyposmotic medium increased 86 Rb efflux rate from 0.015 min ุ1 to a maximal of 0.049 min ุ1 and a net release of 30%. This osmosensitive efflux was inhibited by Ba 2ุ (0.028 min ุ1 ), quinidine (0.024 min ุ1 ), and charybdotoxin (0.040 min ุ1 ), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca 2ุ from the hyposmotic medium increased 86 Rb efflux to a maximal rate constant of 0.056 min ุ1 and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca 2ุ removal in isosmotic medium. This isosmotic 86 Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba 2ุ , nitrendipine, and Mg 2ุ . With the swellinginduced [Ca 2ุ ] i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic 86 Rb was 30% reduced. The Ca 2ุ entry blockers Cd 2ุ , Ni 2ุ , La 3ุ , and Gd 3ุ did not affect 86 Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca 2ุ , because these agents did not affect the [Ca 2ุ ] i rise and the inhibition persisted in the absence of external Ca 2ุ . The phospholipase C blocker U-73122 did not affect [Ca 2ุ ] i nor 86 Rb efflux. Blockers of Ca 2ุ /calmodulin W7 and KN-93 decreased 86 Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated 86 Rb release in hyposmotic conditions only when [Ca 2ุ ] i was raised to about 1 M, suggesting the implication of maxi-K ุ channels at this [Ca 2ุ ] i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that 86 Rb efflux in cerebellar astrocytes is largely (70%) Ca 2ุ -independent and the Ca 2ุ -dependent fraction is sustained essentially by Ca 2ุ released from the endoplasmic reticulum and mediated by a mechanism involving Ca 2ุ /calmodulin.