Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl current, I_Cl(vol), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl equilibrium potential. The sequence of anion permeability was I > Br > Cl > gluconate. The Cl channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I_Cl(vol) was cell cycle dependent, being high in G₁ phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl channel blockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I_Cl(vol) by NPPB (100 µM) arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl current associated with RVD may therefore play an important role during the cell cycle progress.