It is well established that the release of vasodilators and vasoconstrictors from vascular endothelium regulates vascular smooth muscle contraction. In this report, we investigate the role of the endothelium in the development and maintenance of constitutive vascular contractility. For that purpose, contractile activity of cultured bovine aortic smooth muscle cells (BASMCs) embedded in collagen gels was monitored by changes in gel diameter. After culturing for 5 days, ATP- and high KCl solution-induced contractions were significantly enhanced in the gels that were overlaid with bovine aortic endothelial cells (BAECs) or were cultured with conditioned medium of cultured BAECs. ATP-induced Ca²⁺ transients, recorded in BASMCs cultured with conditioned medium of BAECs, were markedly augmented, but high KCl-induced Ca²⁺ transients were not affected. BASMCs in control gels were spindle shaped, and those in endothelium-treated gels were more elongated and interconnected. The endothelial conditioned medium also strongly affected the intracellular distribution of actin fibers. Conditioned medium of BAECs contained TGFβ₁ and TGFβ₂. The TGFβ receptor antagonist SB431542 as well as simultaneous treatment with TGFβ₁ and TGFβ₂ neutralizing antibodies completely reversed the above effects of endothelial conditioned medium on BASMCs. BAECs medium induced phosphorylation of Smad2 and increased ATP-induced phosphorylation of myosin light chain in BASMCs. The present results indicate that the release of TGFβ₁ and TGFβ₂ from vascular endothelium affects the contractility of vascular smooth muscle cells by altering their morphology and agonist-induced Ca²⁺ mobilization.