Bovine aortic endothelial and smooth muscle cells in culture were incubated with arachidonic acid or prostaglandin H2. The amount of prostacyclin nd thromboxane A2 synthesized ws then determined by specific radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2. Although smooth muscle cells produced only 6-keto-prostaglandin F1 alpha and thromboxane B2 in a ratio of 5:1 to 10:1. The same ratio of these metabolites of arachidonic acid ws also found when prostaglandin production from endogenous arachidonic acid was stimulated in endothelial cells by the ionophore A23187. Cyclooxygenase inhibitors inhibited the production of both metabolites equally, whereas thromboxane synthetase inhibitors selectively inhibited the production of thromboxane B2. Cells in culture were also incubated with [1-14C]arachidonic acid and the extracted products were identified by two-dimensional thin-layer chromatography. 6-Keto-prostaglandin F1 alpha was the only metabolite produced by smooth muscle cells, but endothelial cells synthesized 6-keto prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, and prostaglandin F2 alpha.