Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH₂-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC-α inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 μM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin.