Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause K⁺, Cl^- and H₂O effluxes, leading to cell shrinkage; however, the physiological significance of RVD is unclear. Recently, we showed that hypotonicity stimulated transepithelial Na⁺ reabsorption via elevation of epithelial Na⁺ channel (ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner, and that reduction of intracellular Cl^- concentration ([Cl^-]_i) stimulated the Na⁺ reabsorption. These suggest that RVD would reveal its stimulatory action on the Na⁺ reabsorption by reducing [Cl^-]_i. However, the reduction of [Cl^-]_i during RVD has not been definitely analyzed due to technical difficulties involved in halide-selective fluorescent dyes. In the present study, we have developed a new method for the measurement of [Cl^-]_i change during RVD by using a high-resolution flow cytometer with a halide-sensitive fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester (MQAE). The [Cl^-]_i in A6 cells in an isotonic medium was 43.6 ± 3.1 mM. After hypotonic shock (268 to 134 mOsm/kg H₂O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl^-]_i from 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl^- channel blocker) or quinine (K⁺ channel blocker), we did not detect the reduction of [Cl^-]_i. Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl^-]_i, and that RVD shows its action via reduction of [Cl^-]_i acting as an intracellular signal regulating cellular physiological functions.