Moesin is a member of the ERM family, a family of cross linkers between the plasma membrane and the actin cytoskeleton, which are known to be activated by phosphorylation. Previously, we reported the RhoA and Rho kinase-dependent phosphorylation of moesin at Thr558 in hippocampal neuronal cells by glutamate. Here we studied the induction of moesin phosphorylation by KCl (60mM) in PC12 cells. Moesin phosphorylation at Thr558 was increased after 2 min of KCl treatment, peaked at 5 min and returned to the basal level by 60 min. KCl also activated Rac1, but not RhoA, in PC12 cells, and KCl-induced moesin phosphorylation was suppressed in dominant negative Rac1 (N17 Rac1) expressed cells. The inhibition of PKA, known as an upstream kinase of Rac1, abolished Rac1 activation and moesin phosphorylation by KCl. Interestingly, the phosphorylation of moesin by KCl was independent of KCl-induced membrane depolarization and calcium influx, but was dependent on KCl-induced chloride conductance. 60 mM KCl induced chloride conductance in PC12 cells and pretreatment with Cl- channel blocker abolished Rac1 activation and moesin phosphorylation by KCl. These results suggest that the phosphorylation of moesin at Thr558 in PC12 cells by KCl treatment is PKA and Rac1 dependent, and that KCl-induced chloride conductance is involved in the activation of this signaling system.