Computer-aided time lapse fluorescence videomicroscopy was used to study single cell migration behavior of human aortic endothelial cells on fibronectin coated substrates of varying protein surface density. The role of receptors α5β1, αvβ3, and α4β1 in mediating cell adhesion and migration on fibronectin was characterized using integrin specific monoclonal antibodies. Matrix density had a direct effect on controlling the proportion of migrating cells and the directional persistence of cell movement (p < 0.01). While there was relatively little influence of fibronectin surface density on absolute migration speed, the ability of endothelial cells to disperse over a surface, as measured by the dispersion coefficient, was biphasic with respect to the surface density of this matrix protein (p < 0.005). Both cell speed and the proportion of migrating cells was controlled by α4β1 (p < 0.01). However, α5β1 selectively regulated the transformation of stationary cells to those exhibiting motile behavior (p < 0.05). Migratory responses on fibronectin were not influenced by blockade of the αvβ3 receptor. It is noteworthy that cell surface adhesive receptors which control commitment to a motile phenotype are not necessarily the same as those that control migration speed.