When bovine pulmonary artery endothelial cells are cultured in a medium supplemented with linoleic acid, their capacity to produce prostacyclin (PGI2) is reduced by about 60%. This reduction occurs when PGI2 formation is stimulated by the addition of either the calcium ionophore A23187 or arachidonic acid. In addition, supplementation with linoleic acid reduced the production of prostaglandin E2 and F2 alpha from 1-14C-arachidonic acid by more than 50%. The capacity of cultured bovine pulmonary vein and aortic endothelial cells to convert extracellular arachidonic acid into PGI2 also was reduced by about 50% when the growth medium was supplemented with linoleic acid. Although bovine pulmonary artery endothelial cells incorporated large amounts of 1-14C-linoleic acid into cellular phospholipids and triglycerides, a maximum of only 2.3% of the radioactivity was converted to arachidonic acid in 24 hours. The most prevalent radioactive metabolite was eicosadienoic acid, the elongation product of linoleic acid. As compared with linoleic acid, the bovine endothelial cells incorporated 30% more 1-14C-arachidonic acid into phospholipids and 60% more into triglycerides. When the growth medium was supplemented with linoleic acid, the percentage of this fatty acid in cellular lipids increased 3- to 4.5-fold and eicosadienoic acid accumulated, accounting for up to 9% of the cellular fatty acids. This increase was accompanied by a 30% to 45% reduction in arachidonic acid. These findings, together with our previous results with human umbilical vein endothelium, suggest that an inability to convert large amounts of linoleic to arachidonic acid and a suppressive effect of linoleic acid enrichment on prostaglandin production may be general properties of endothelial cells.