3T3 cells that have undergone adipose differentiation in vitro secrete factor(s) that stimulate angiogenesis (neovascularization) in vivo. When medium containing 0.5% fetal calf serum was conditioned by 3T3-F442A adipocytes, it stimulated angiogenesis when placed on the chicken chorioallantoic membrane. Control medium or medium conditioned by preadipocytes did not stimulate angiogenesis, even at much higher doses. Thus, the production of the angiogenic activity is strongly dependent upon differentiation of the adipocytes. The degree of the angiogenic response to adipocyte-conditioned medium was potentiated by heparin; heparin added to unconditioned medium or to preadipocyte-conditioned medium was not angiogenic. The adipocyte-conditioned medium also strongly stimulated, in a differentiation-dependent fashion, the motility of aortic and capillary endothelial cells in a modified Boyden chamber assay. Checkerboard analysis cells indicated that 75% of the motility-stimulating activity was chemotactic in nature. The chemotactic activity has an apparent specificity for endothelial cells, in that chemotaxis of smooth muscle cells and fibroblasts was stimulated to a much lesser extent. These results, in conjunction with our previous demonstration of an endothelial cell mitogen produced by 3T3 adipocytes [Castellot, J. J., Jr., Karnovsky, M. J. & Spiegelman, B. M. (1980) Proc. Natl. Acad. Sci. USA 77, 6007-6011] indicate that the differentiation of these cells is closely linked to the production of factors that stimulate angiogenesis in vivo and growth and chemotaxis of endothelial cells in vitro.