Adenoviral vectors are known to transduce hepatocytes in normal liver tissue with high efficiency. The aim of this study was to investigate whether sinusoidal endothelial cells, which separate hepatocytes from the bloodstream in the sinusoidal lumen, are permissive for infection by adenoviruses. We show here that microvascular liver sinusoidal endothelial cells are not infected by adenovirus type 5 in vivo or in vitro unless high MOIs are used. In contrast, macrovascular endothelial cells from aorta are efficiently infected by adenovirus type 5. In addition, Kupffer cells, similar to sinusoidal endothelial cells, are not infected by adenovirus type 5. Liver sinusoidal endothelial cells do not express the integrin receptor alpha(v)beta3, which is required for efficient infection by adenoviruses. Our results demonstrate that hepatocytes are the main cell population of the liver that is infected by adenovirus type 5.