During septic states, the lungs produce increased amounts of glutamine, an event that is mediated by both endotoxin and the glucocorticoid hormones and is presumed to be due to accelerated intracellular glutamine biosynthesis. Because enhanced net glutamine release in vivo could also be due to a decrease in cellular uptake, we assayed glutamine transport in cultured rat microvascular pulmonary endothelial cells. The effect of Escherichia coli endotoxin (LPS, 1 μg/mL), various cytokines, and dexamethasone (DEX, 0.1 μmol/L) on glutamine transport activity was studied in rat lung microvascular endothelial cells grown in varying glutamine concentrations (0, 0.1, 0.5, and 2 mmol/L). Experiments were also performed in cells treated with cycloheximide, actinomycin D, or chelerythrine chloride. More than 90% of glutamine transport was mediated by the Na⁺‐dependent transport system ASC. DEX and LPS inhibited endothelial glutamine uptake in a time‐ and dose‐dependent manner, a response that was only observed when incubation medium contained the lower concentrations of glutamine. Neither DEX nor LPS altered transport activity in cells cultured in medium containing 2 mmol glutamine/L. There was no synergistic or additive effect when both compounds were added together. The cytokines tumor necrosis factor α, interleukin (IL) 1, IL‐2, and IL‐6 did not alter glutamine transport. Both DEX and LPS inhibited glutamine transport by decreasing transporter maximal transport velocity (V_max) without affecting transporter affinity (K_m). Cycloheximide and actinomycin D abrogated the inhibition of transport activity that was observed in DEX‐ or LPStreated cells, whereas the protein kinase C inhibitor chelerythrine chloride had no effect on either control or stimulated glutamine transport. These data suggest that DEX and LPS “down‐regulate” glutamine uptake by lung microvascular endothelial cells by inducing the synthesis of an inhibitory protein that modulates the activity of the System ASC protein. This response in vitro appears to be influenced by the extracellular glutamine concentration. This decrease in microvascular endothelial glutamine transport may be one mechanism by which net lung glutamine release is enhanced during critical illness. (Journal of Parenteral and Enteral Nutrition 19:477–481, 1995)