Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.