In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na⁺ transport. Hypotonic action on Na⁺ transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na⁺ transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial Na⁺ channel (ENaC), increasing Na⁺ transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca²⁺ in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca²⁺ concentration in A6 cells, we hypothesized that Ca²⁺-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca²⁺-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na⁺ transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca²⁺ plays a role as the second messenger in hypotonic stress-induced Na⁺ transport by stimulating transcription of SGK1 and ENaC subunits.