Endothelial K+ and Ca2+ homeostasis plays an important role in the regulation of tissue supply and metabolism under normal and pathological conditions. However, the exact molecular mechanism of how Ca2+ is involved in the regulation of K+ homeostasis in capillary endothelial cells, especially under oxidative stress, is not clear. To reveal Ca2+-triggered pathways, which modulate K+ homeostasis, Ca2+/calmodulin-dependent protein kinase II and voltage-gated outward K+ currents were studied in rat brain capillary endothelial cells under hypoxia. Whole cell voltage-clamp measurements showed voltage-gated outward K+ current with transient and sustained components. mRNA and protein of Ca2+/calmodulin-dependent protein kinase II delta2 and two gamma isoenzymes were identified. Activation of the isoforms (autophosphorylation) was typically achieved by the Ca2+ ionophore ionomycin, which was prevented by the Ca2+/calmodulin-dependent protein kinase II-specific inhibitor KN-93. Hypoxia resulted in autophosphorylation of the delta2 and gammaB isoforms, augmented the current amplitude, increased the inactivation time constant, and decreased the extent of inactivation of the transient current. KN-93 prevented both the activation of the isoforms and the alterations in the K+ current characteristics. It is concluded that the activation of Ca2+/calmodulin-dependent protein kinase II decreases inactivation of the voltage-gated outward K+ current, thereby counteracting depolarization of the hypoxic endothelium.