The transport characteristics of l- and d-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). l-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate ofl-histidine. Ouabain, an inhibitor of Na+-K+-ATPase, also reduced uptake ofl-histidine. Moreover, the initial l-histidine uptake rate was reduced by the substitution of Na+ with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, l-glutamic acid γ-monohydroxamate, also inhibited uptake of l-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that l-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na+. Moreover, the Na+-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on l-histidine uptake in Na+ removal, indicating facilitated diffusion by a Na+-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for l-histidine uptake into rat LMECs, a Na+-dependent system N and Na+-independent system L process. On the other hand, the uptake of d-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na+replacement. Although the uptake of d-histidine was reduced in the presence of BCH, the addition of l-glutamic acid γ-monohydroxamate did not significantly decrease uptake ofd-histidine. These results suggest that the uptake ofd-histidine by rat LMECs has different characteristics compared with its isomer, l-histidine, indicating that system N transport did not involve d-histidine uptake.