Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.