Endoglin is a membrane glycoprotein that plays an important role in cardiovascular development and angiogenesis. We examined the role of endoglin in the control of vascular tone by measuring nitric oxide (NO)-dependent vasodilation in haploinsufficient mice (Eng+/-) and their Eng+/+ littermates. The vasodilatory effect of acetylcholine, bradykinin, and sodium nitroprusside was assessed in anesthetized mice; in isolated, perfused hindlimbs; and in aortic rings. The substantial hypotensive and vasodilatory response induced by acetylcholine and bradykinin in Eng+/+ was markedly reduced in Eng+/- mice. Both kinds of animals had similar responses to sodium nitroprusside, suggesting that the deficient vasodilatory effect is not due to a NO response impairment. Urinary and plasma concentrations of nitrites, a NO metabolite, were lower in Eng+/- than in Eng+/+ mice. The levels of endothelial nitric oxide synthase (eNOS) in kidneys and femoral arteries were about half in Eng+/- than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/- compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively. Thus, our results in vivo and in vitro demonstrate a relationship between endoglin and NO-dependent vasodilation mediated by the regulation of eNOS expression.