Despite the central role of the liver in glutamine homeostasis in health and disease, little is known about the mechanism by which this amino acid is transported into sinusoidal endothelial cells, the second most abundant hepatic cell type. To address this issue, the transport of L-glutamine was functionally characterized in hepatic endothelial cells isolated from male rats. On the basis of functional analyses, including kinetics, cation substitution, and amino acid inhibition, it was determined that a Na+-dependent carrier distinct from system N in parenchymal cells, with properties of system ASC or B0, mediated the majority of glutamine transport in hepatic endothelial cells. These results were supported by Northern blot analyses that showed expression of the ATB0 transporter gene in endothelial but not parenchymal cells. Concurrently, it was determined that, whereas both cell types express glutamine synthetase, hepatic endothelial cells express the kidney-type glutaminase isozyme in contrast to the liver-type isozyme in parenchymal cells. This represents the first report of ATB0 and kidney-type glutaminase isozyme expression in the liver, observations that have implications for roles of specific cell types in hepatic glutamine homeostasis in health and disease.