Endothelin is a 21 amino acid peptide secreted by endothelial cells and is the most potent vasoconstrictor known. The present study examines regulatory mechanisms of endothelin secretion, focusing on the role of protein phosphorylation. Endothelin secretion was measured by radioimmunoassay in primary cultures of human umbilical vein endothelial cells. While treatment that raised cAMP levels reduced the basal endothelin secretion rate, agents that elevated cGMP had no effect. Downregulation or inhibition of protein kinase C resulted in decreased endothelin secretion, suggesting that protein kinase C regulates endothelin secretion in the opposite direction to cAMP dependent protein kinases Okadaic acid, at concentrations that selectively inhibit protein phosphatases 2A, reduced the endothelin secretion and the effects of okadaic acid and db‐cAMP were additive. Endothelin production was stimulated by fetal calf serum and by the protein kinase inhibitor 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine (H7), but was inhibited by the calmodulin antagonist trifluoperazine. The present findings that regulators of cAMP‐dependent protein kinases, protein kinase C, calmodulin, and protein phosphatase 2A all affect endothelin secretion suggest that endothelin secretion is controlled by phosphorylation/dephosphorylation of as yet unidentified regulatory proteins within the cell. © 1994 Wiley‐Liss, Inc.