The Na+/K+/2Cl(-) cotransporter (NKCC) mediates electroneutral transport of 2Cl(-) coupled with Na+ and K+ across the plasma membrane, and plays crucial roles in Cl- uptake into the cells, homeostasis of cellular Cl-, and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCCl (an isoform, of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCCl protein was increased by NGF treatment. Knock-down of NKCCl by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCCl into cells for clarification of intracellular localization of NKCCl revealed that the EGFP-rNKCCl was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCCl plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells. (c) 2007 Elsevier Inc. All rights reserved.