Background. Angiogenesis is critical to tumor growth. Gax, a homeobox transcription factor whose expression in the adult is restricted mainly to the cardiovascular system, strongly inhibits growth factor-stimulated phenotypic modulation of vascular smooth muscle cells in vitro and in vivo. The function of Gax in vascular endothelium is unknown, but we hypothesized that it may play a similar role there. We therefore studied Gax expression in vascular endothelial cells and its effects on proliferation and tube formation. Materials and methods. Gax expression in normal endothelial cells was examined in vitro by Northern blot and reverse transcriptase polymerase chain reaction and in vivo by immunohistochemistry. A replication-deficient adenovirus was then used to express Gax in human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, 3H-thymidine uptake, p21 expression, and tube formation on reconstituted basement membrane were measured at different viral multiplicities of infection. Results. Gax mRNA was detected in HUVECs by reverse transcriptase polymerase chain reaction and Northern blot analysis and in normal vascular endothelium by immunohistochemistry. Compared with controls transduced with a virus expressing β-galactosidase, Gax strongly inhibited HUVEC proliferation and mitogen-stimulated 3H-thymidine uptake. p21 expression in HUVECs transduced with Gax was increased up to 5-fold as measured by Northern blot, and p21 promoter activity was activated by 4- to 5-fold. Tube formation on Matrigel was strongly inhibited by Gax expression. Conclusions. Gax is expressed in vascular endothelium and strongly inhibits endothelial cell activation in response to growth factors and tube formation in vitro. These observations suggest that Gax inhibits endothelial cell transition to the angiogenic phenotype in response to proangiogenic growth factors and, as a negative regulator of angiogenesis, may represent a target for the antiangiogenic therapy of cancer.