Although endothelin-1-stimulated contractile force generation by stellate cells is believed to play an important role in hepatic pathophysiology, the molecular signals that mediate this process are incompletely understood. The aim of this study was to test the hypothesis that myosin mediates the contractile force generated by stellate cells in response to endothelin-1. Contractile force generation by primary and immortalized stellate cells was directly and quantitatively measured. Myosin phosphorylation and reorganization, and actin stress fiber formation were investigated in immortalized stellate cells. Endothelin-1 stimulated a rapid and robust generation of contractile force by primary and immortalized stellate cells with a similar dose dependence. Myosin phosphorylation, actin stress fiber assembly, and reorganization of myosin to stress fibers were induced by concentrations of endothelin-1 that also stimulated stellate cell contraction. BQ-123, a selective endothelin receptor antagonist, inhibited myosin phosphorylation and contractile force generation. Y-27632, which selectively inhibits rho-associated kinase, also blocked endothelin-1-stimulated myosin phosphorylation and contractile force generation with a similar dose dependence. These results suggest that endothelin-1-stimulated contractile force generation by stellate cells is mediated by myosin.