Endoglin, a transforming growth factor beta (TGF-beta) co-receptor, modulates TGF-beta responses, which in endothelial cells are mediated via ALK5 and ALK1 pathways. Since mutations in the ENG or ALK1 genes are associated with hereditary hemorrhagic telangiectasia, we hypothesized that endoglin preferentially modulates the ALK1 pathway. We studied the expression of downstream target genes of both pathways in endothelial cells derived from Endoglin null (Eng -/-) and control (Eng+/+) embryos, and showed that Eng-/- cells express higher basal levels of ALK1 regulated Smad6, Smad7, and Id1 mRNA. Transfection experiments using ALKI and ALK5 reporter genes confirmed increases in ALK1 mediated TGF-beta responses. Higher levels of ALK5 were observed, supporting the concept that the ALK1 receptor complex requires ALK5 for activity. Our data implicate endoglin as a critical player in maintaining the balance of TGF-beta pathways in endothelial cells and vascular homeostasis.