ABSTRACT— To determine whether von Willebrand Factor (vWF) is a valid marker of liver endothelial cells, we determined vWF immunoreactivity in rat and guinea pig liver sections and in smears of elutriated nonparenchymal cells isolated from these two species. In frozen sections, positive staining for vWF was seen only in the endothelium lining large hepatic vessels in both species, and no immunoactivity was detected in the sinusoids. On the other hand, immunohistochemical staining for vimentin (a marker of mesenchymal cells) showed positive reaction throughout the vascular and sinusoidal endothelial cells in both the rat and guinea pig liver. In fractions of elutriated rat and guinea pig nonparenchymal liver cells, which included almost exclusively liver endothelial cells, only 25–40% of the cells displayed a positive reaction for vWF. However, when these same fractions were stained for vimentin, 70–95% of the cells exhibited immunoreactivity. Most of the vWF-negative cells were not red and white blood cells, biliary epithelial and Kupffer cells, and hepatocytes, and had ultrastructural features of sinusoidal endothelial cells. We conclude that in both the rat and guinea pig, liver sinusoidal endothelial cells do not exhibit vWF immunoreactivity. Thus, in routine immunohistochemical assays, vWF is not an accurate marker of rat and guinea pig liver endothelial cells. Vimentin is more appropriate for this purpose, provided that other mesenchymal cells are separated or independently identified.