Recent studies suggest that chloride (Cl-) channels regulate tumor cell migration. In this report, we have used antisense oligonucleotides specific for ClC-3, the most likely molecular candidate for the volume-activated Cl- channel, to investigate the role of ClC-3 in the migration of nasopharyngeal carcinoma cells (CNE-2Z) in vitro. We found that suppression of ClC-3 expression inhibited the migration of CNE-2Z cells in a concentration-dependent manner. Whole-cell patch-clamp recordings and image analysis further demonstrated that ClC-3 suppression inhibited the volume-activated Cl- current (I(Cl,vol)) and regulatory volume decrease (RVD) of CNE-2Z cells. The expression of ClC-3 positively correlated with cell migration, I(Cl,vol) and RVD. These results strongly suggest that ClC-3 is a component or regulator of the volume-activated Cl- channel. ClC-3 may regulate CNE-2Z cell migration by modulating cell volume. ClC-3 may be a new target for cancer therapies.