Cell spreading and migration involve both surface attachment and cytoskeletal reorganization, leading to membrane extension and ultimately cell polarization, processes that are stimulated and coordinated through intracellular signal transduction pathways mediated by adhesion and chemoattractant receptors. In addition, the rigidity of the substratum influences cell polarization, membrane protrusion and ultimately cell migration. In fibroblasts and many other cells, activation of the phosphoinositide (PI) 3-kinase pathway is strictly required for motility. PI 3-kinases generate specific 3' PI lipid products, which act as membrane second messengers, and the spatial pattern of 3' PI density in the membrane is thought to control the directionality of membrane protrusion and cell migration. As a model system, we have quantitatively followed the time course of PI 3-kinase activation, membrane spreading and post-spreading motility of mouse fibroblasts following their initial attachment to fibronectin- and poly-L-lysine-coated substrates. Using total internal reflection fluorescence microscopy in conjunction with fluorescent 3' PI probes, we have observed activation of PI 3-kinase signaling during cell spreading that is independent of surface coating. In an attempt to further elucidate this unique mechanism for PI 3-kinase activation, we perturb both substrate rigidity and intracellular elements that regulate membrane protrusion. We report that the extent of PI 3-kinase activation is a function of surface rigidity, and that Rho family GTPases and the cytoskeleton are important mediators of PI 3-kinase activation during spreading. These results suggest that fibroblasts integrate mechanical and chemical stimuli at the level of PI 3-kinase signaling.