Expression of vascular endothelial growth factor (VEGF) is tightly regulated to achieve normal angiogenesis. The objective was to examine regulation of VEGF by the activin-like kinase receptors (ALKs) ALK1 and ALK5. Transforming growth factor beta1 (TGFbeta1) and bone morphogenetic protein-9 (BMP-9) enhanced and suppressed VEGF expression, respectively, in aortic endothelial cells, as determined by real-time polymerase chain reaction, immunoblotting, cell proliferation, and tube formation. The use of small interfering RNA revealed that TGFbeta1 stimulated VEGF expression by activating ALK5, TGFbeta type II receptor, and SMAD2, whereas BMP-9 suppressed it by activating ALK1, BMP type II receptor, and SMAD1. ALK1 signaling occurred independently of ALK5 activity. Partial ALK1 deficiency in vitro and in vivo resulted in elevated VEGF expression. In vitro, increased BMP-9 levels normalized VEGF expression in cells with partial, but not severe, ALK1 deficiency. Time course experiments revealed that an increase in ALK1 expression induced by BMP-4, an angiogenic stimulus, preceded induction of ALK5 and VEGF in control cells. In ALK1-deficient cells, however, VEGF expression occurred earlier and was abnormally high, even though ALK5 was not induced. Our results suggest that ALK1 and ALK5 are both essential for correct regulation of VEGF, and that disruption of either pathway leads to disease.