Background: Transforming growth factor-β (TGF-β) isoforms have been implicated as both pro- and anti-angiogenic modulators. In this study we addressed the roles of TGF-β isoforms on coronary tubulogenesis. Methods: Embryonic (E6) quail ventricular specimens were explanted onto collagen gels allowing endothelial cells to migrate and form vascular tubes. Growth factors and/or neutralizing growth factor antibodies were added to the cultures. Endothelial cells were identified using a quail endothelial cell marker, QH1. Image analysis was used to quantify aggregate tube length. Results: Addition of any isoform (TGF-β<sub>1</sub>, TGF-β<sub>2</sub> or TGF-β<sub>3</sub>) virtually prevented tubulogenesis (>95% inhibition), while stimulation of tubulogenesis occurred by adding neutralizing antibodies to TGF-β<sub>3</sub>, but not to TGF-β<sub>1</sub> or -β<sub>2</sub>. When all three isoforms were added, tubulogenesis was enhanced, indicating the key role of TGF-β<sub>3</sub>. Documentation of the inhibitory effect of TGF-β isoforms on tubulogenesis is further supported by our experiments in which the marked enhancement of tube formation by bFGF and VEGF was negated when exogenous TGF-β<sub>1</sub>, -β<sub>2</sub>, or -β<sub>3</sub> were added to the cultures. Conclusions: (1) TGF-β<sub>1</sub>, -β<sub>2</sub> and -β<sub>3</sub> each inhibits angiogenesis; (2) cooperation between the three TGF-β isoforms and other angiogenic factors is essential for the regulation of normal tubulogenesis and (3) the stimulatory effect of VEGF or bFGF on tubulogenesis is negated by exogenous TGB-βs.