The study investigates the regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. Soluble factors like lysophosphatidic acid (LPA) are known to activate Rho, but the role of cell adhesion to extracellular matrices (ECM) in Rho activation remains controversial. The authors developed an assay to measure GTP-bound Rho and found that plating Swiss 3T3 cells on fibronectin-coated dishes initially inhibited Rho activity, followed by a transient activation phase enhanced by serum. LPA induced transient Rho activation in serum-starved cells, while in suspended cells, Rho activation was sustained. Higher Rho activity was observed in suspended cells compared to adherent cells in the presence of serum, indicating an adhesion-dependent negative feedback loop. Additionally, cytochalasin D and colchicine, which disrupt the actin cytoskeleton, also activated Rho, suggesting that Rho activation is not solely dependent on cytoskeletal tension. These findings highlight the complex regulation of Rho by soluble factors, ECM proteins, and cytoskeletal structures, and suggest that Rho plays a crucial role in maintaining homeostasis and preventing excessive contraction under physiological conditions.The study investigates the regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. Soluble factors like lysophosphatidic acid (LPA) are known to activate Rho, but the role of cell adhesion to extracellular matrices (ECM) in Rho activation remains controversial. The authors developed an assay to measure GTP-bound Rho and found that plating Swiss 3T3 cells on fibronectin-coated dishes initially inhibited Rho activity, followed by a transient activation phase enhanced by serum. LPA induced transient Rho activation in serum-starved cells, while in suspended cells, Rho activation was sustained. Higher Rho activity was observed in suspended cells compared to adherent cells in the presence of serum, indicating an adhesion-dependent negative feedback loop. Additionally, cytochalasin D and colchicine, which disrupt the actin cytoskeleton, also activated Rho, suggesting that Rho activation is not solely dependent on cytoskeletal tension. These findings highlight the complex regulation of Rho by soluble factors, ECM proteins, and cytoskeletal structures, and suggest that Rho plays a crucial role in maintaining homeostasis and preventing excessive contraction under physiological conditions.