Studies were performed to characterize the identity of the factors present in calf serum that stimulate endothelin (ET) production by vascular endothelial cells in culture. Two putative candidates responsible for the ET-releasing activity present in serum are members of the transforming growth factor-beta (TGF beta) superfamily, TGF beta 1 and activin A. TGF beta 1 antiserum was used to block the actions of TGF beta 1, and follistatin was used to block the actions of activin. Treatment of serum with TGF beta 1 antiserum attenuates ET production by vascular endothelial cells. TGF beta 1 antiserum (1 microliters) completely prevents the release of ET produced by 0.1% and 0.3% serum. Follistatin is a potent inhibitor of activin-induced ET release but does not alter serum-induced ET production. Because serum-induced ET release is dependent on TGF beta, it may be a physiological regulator of ET production by vascular endothelial cells.