AbstractNa+ transport via epithelial Na+ channel (ENaC) occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. In this study, we have examined the effects of cytosolic Na+ and Cl− on ENaC activity by patch clamping single channel recording method in mouse cortical collecting duct cells (M1). Cytosolic Na+ exerts its effect in change of ENaC open probability (Po). High cytosolic Na+ significantly reduces ENaC Po. No change in channel conductance by cytosolic Na+ is observed. However, decrease of cytosolic Cl− concentration significantly increases channel conductance and ENaC Po. This effect is due to the right shift of ENaC I–V curve to positive membrane potential. The virtue of ENaC conductance remains the same. Cl− channels like CFTR and VRAC are unlikely to be involved in this regulation. The results suggest that cytosolic Cl− could serve as a mediator to regulate ENaC activity, in accordance with the activities of Cl− channels. J. Cell. Physiol. 216: 453–457, 2008. © 2008 Wiley‐Liss, Inc.