We investigated the effect of inhibiting Na⁺-K⁺-ATPase on the basolateral 18-pS K⁺ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na⁺-K⁺-ATPase with strophanthidin decreased the activity of the 18-pS K⁺ channel and increased the intracellular Ca²⁺ to 420 nM. Removal of extracellular Ca²⁺ abolished the effect of strophanthidin. When intracellular Ca²⁺ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K⁺ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca²⁺-induced inhibition, the effect of 400 nM Ca²⁺ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca²⁺. This suggested that the inhibitory effect of high concentrations of Ca²⁺ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca²⁺ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca²⁺ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N-nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N-nitro-L-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca²⁺ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na⁺-K⁺-ATPase is mediated by intracellular Ca²⁺ and the inhibitory effect of high concentrations of Ca²⁺ is the result of interaction of nitric oxide with superoxide.