Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been clearly elucidated. Here we show that plasma membrane depolarization provoked by distinct means including high extracellular K⁺, the lipophilic cation tetraphenylphosphonium or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca²⁺-signal, and removal of external Ca²⁺ rather promoted than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca²⁺ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator since: a) depolarization stimulated Rho and induced its peripheral translocation; b) inhibition of Rho by Clostridium Difficile Toxin B or C3 transferase abolished MLC phosphorylation; and c) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiologic depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na⁺-alanine cotransporter stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na⁺-dependent manner. Together our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization-triggered, Rho-ROK-mediated but Ca2⁺ signal-independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport.