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 of L-histidine. Ouabain, an inhibitor of Na⁺-K⁺-ATPase, also reduced uptake of L-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 -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 -monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.