We have determined the chemotactic response of bovine aortic endothelial cells to fibronectin and to endothelial cell mitogens (endothelial cell growth supplement, tumor extract), using blind-well chemotaxis chambers. Fibronectin induced a chemotactic response in bovine aortic endothelial cells; at 100 micrograms/ml, chemotaxis increased by 440% above that observed in negative controls (p less than 0.001). The chemotactic response plateaued with time, paralleling the disappearance of the fibronectin concentration gradient in the chambers. As further evidence that chemotaxis was measured, we observed that cell migration did not occur when cells were incubated with fibronectin in the absence of a concentration gradient. Endothelial cell mitogens increased bovine aortic endothelial cell proliferation in our experiments but did not stimulate chemotaxis above background levels. In contrast, fibronectin inhibited cell proliferation by 23%. We present a hypothetical model of the role of fibronectin in evoking endothelial cell chemotaxis during tumor neovascularization.