In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na⁺ transport. Hypotonic action on the Na⁺ transport is composed of two phases, a non-genomic early phase and a genomic delayed phase. Although previous works reported that during the genomic phase hypotonic stress stimulates the transcription of Na⁺ transport-related genes, such as SGK1 and subunits of epithelial sodium channel (ENaC), increasing the Na⁺ transport, the mechanism is still unknown. We focused the present study on the role of the intracellular Ca²⁺ in the hypotonicity-induced SGK1 and ENaC subunits transcription. Since hypotonic stress raises the intracellular Ca²⁺ concentration in A6 cells, we hypothesized that Ca²⁺-dependent signals participate in the genomic action. Using real-time QRT-PCR and western blotting 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 the 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 without any effect on the early phase. These observations indicate for the first time that the intracellular Ca²⁺ plays a role as the second messenger in the hypotonic stress-induced Na⁺ transport by stimulating the transcription of SGK1 and ENaC subunits.