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This study aimed to investigate the acute and chronic effect of methotrexate on the intestinal substance P metabolism after a single administration to rats. Methotrexate caused a significant increase in the number of substance P-containing cells in the ileal mucosa both at 24 and 96 h. Most of enterochromaffin cells expressing l-tryptophan hydroxylase contained substance P. The expression of Tac1 mRNA was increased by methotrexate at 24 h, but not at 96 h. Thus, methotrexate causes acute hyperplasia of enterochromaffin cells in the intestinal mucosa of rats with a transient increase in the production of substance P.