Human endothelial cells in culture synthesize prostaglandins and release these products into the culture medium. The major products of arachidonic acid metabolism were identified by high pressure liquid chromatography or thin layer chromatography, and release of prostaglandins was measured by radioimmunoassays. Addition of histamine or bradykinin enhanced release of prostaglandins in both arterial and venous endothelial cells. Other vasoactive compounds including angiotensin II, vasopressin, substance P, epinephrine, norepinephrine, or isoproterenol were ineffective. Release of prostaglandins by histamine was concentration-related, and involved H1 receptors, as determined by addition of histamine antagonists. Incubation of endothelial cells with 14C-arachidonic acid resulted in a time-dependent uptake into cell lipids, where most of the radioactivity was incorporated into phosphatidyl choline and neutral lipids. Endothelial cells released 14C-arachidonic acid as well as 14C-prostaglandins in response to either histamine or bradykinin. The enhanced release of 14C-prostaglandins was inhibited by either indomethacin or mepacrine, but 14C-arachidonic acid release was inhibited only by mepacrine. We conclude that the vasoactive compounds, histamine and bradykinin, stimulate formation of prostaglandins in endothelial cells by the release of arachidonic acid from phospholipids of the cell membrane.