Sinusoidal endothelial cells were isolated from a rat liver by collagenase perfusion and centrifugal elutriation. The cells attached poorly to a plastic dish without coating it even after 24 hr of culture. In the collagen-coated dish, a large number of cells attached to the dish and spread within 24 hr of culture. Centrifugation of the dish immediately after seeding lead to much better attachment of the cells. Fresh rat serum obtained from the portal vein did not increase the attachment, but was essential for the maintenance of the attached cells after the initial 24 hr of culture, when the fetal calf serum was not replaced. The rat serum also induced cell proliferation as determined by thymidine uptake. Its effect was abolished by heattreatment of 56℃ for 30 minutes.