Sinusoidal endothelial cells from adult rat liver have been isolated by centrifugal elutriation and been established in primary culture. Their identification has made use of a novel process of these cells, the sequestration of human acetoacetylated low-density lipoprotein. After its administration in vivo, labeled acetoacetylated low-density lipoprotein within the liver was associated solely with sinusoidal endothelial cells and, when labeled with the stable fluorescent compound 3,3'-dioctadecylindocarbocyanine, provided a means of identifying isolated cells by fluorescence microscopy. In endothelial cell cultures, 90% of the cells were fluorescent and exhibited fenestrae. The collagen phenotype of cultures was assessed by an immunofluorescent approach, which revealed cell-associated type IV collagen only; types I, III, and V were undetectable. In other studies, it was found that these cells lacked factor VIII-R antigen and Weibel-Palade bodies, adding to the evidence that they differ substantially from large-vessel and other capillary endothelia.