Sinusoidal endothelial cells were isolated by collagenase-pronase digestion of rat livers followed by centrifugal elutriation. The main endothelial cell fraction consisted of more than 85% endothelial cells as shown by electron microscopy and enzyme histochemistry. Contamination by Kupffer cells was less than 5%. The endothelial cells formed a coherent stable monolayer on dishes coated with collagen type IV in the presence of an RPMI 1640 medium supplemented with 4% Ultroser. Fc receptors were undetectable immediately after elutriation but reappeared after 12 h in culture. Von Willebrand factor (formerly factor VIII-related antigen) could not be detected unequivocally by immunofluorescence. Unchallenged endothelial cells did not produce eicosanoids. In the presence of free arachidonate, however, prostaglandins D2 and E2 as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha were detected by radioimmunoassay and by high-performance liquid chromatography analysis of [3H]arachidonate-exposed cells. Cells treated with the Ca2+ ionophore A23187 produced the same spectrum of immunologically measured prostanoids. In contrast to Kupffer cells in primary culture, eicosanoid formation by endothelial cells was neither triggered by phagocytotic stimuli nor suppressed by pretreatment with dexamethasone.