Sinusoidal cells isolated from adult rat liver were fractionated by velocity sedimentation at 1 × g (primarly on the basis of size) and the various cell fractions were further analysed by flow cytometry on the basis of forward and perpendicular light scattering and autofluorescence. Cell volume was also measured electronically using a Coulter counter. At least four enriched cell populations were resolved after velocity sedimentation. They corresponded to cells having a modal diameter of 6.5, 7.5, 9, and 11 μm, respectively. Transmission electron microscopy (TEM) analysis of the various cell populations revealed that the 7.5- and 9-μm cell fractions represented two distinct classes of endothelial cells while the 11-μm cells corresponded to Kupffer cells. The 6.5-μm cells were identified as lymphocytes. Fat-storing cells, identified by their autofluorescence and lipid content, were included in the Kupffer population. Further information about the nature of the two physically distinct endothelial cell populations was obtained by TEM. It demonstrated that the smaller endothelial cells possessed quantitatively and relatively less retracted sieve plates than the larger ones. This ultrastructural feature can be possibly correlated to a differential localization of the two classes of endothelial cells within the liver acinus.