In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid co-transport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of co-transporters. Since leukotriene (LTD₄) is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine co-transport by LTD₄. Na-alanine co-transport (ASCT1) was inhibited by LTD₄ in IEC-18 cells. The mechanism of inhibition of ASCT1 by LTD₄ is secondary to a decrease in the affinity of the co-transporter for alanine without a significant change in co-transporter numbers and is not secondary to an alteration in the Na⁺ extruding capacity of the cells. RTQ-PCR and Western blot results indicate that ASCT1 message and protein levels are also unchanged in LTD₄ treated IEC-18 cells. These results indicate that LTD₄ inhibits Na-dependent neutral amino acid co-transport in intestinal epithelial cells. The mechanism of inhibition is secondary to a decrease in the affinity for alanine, which is identical to that seen in villus cells from the chronically inflamed rabbit small intestine where LTD₄ levels are significantly increased.