The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [1-14C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [1-14C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 23%, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D2 (PGD2), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 4-times less eicosanoids (per mg cell protein) than Kupffer cells, and PGD2 was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added 14C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD2 (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostaglandin, PGD2, could stimulate the glucose output in isolated parenchymal cells.