The small intestinal damage induced by the methotrexate (MTX) treatment results in malabsorption and diarrhea. The fluoresceinated methotrexate (F-MTX) may possibly be useful to study such effects of MTX on the small intestine. The purpose of this study is to characterize the transport of F-MTX in the small intestine in order to use it as a membrane transport and cellular marker of MTX. The transport of F-MTX in the rat small intestine (jejunum) was examined in the in vitro everted segments of the intestine. The uptake was pH-dependent and showed a maximal effect at pH 6.0, which was the same as the results of MTX previously reported. Further, it was temperature-dependent and was inhibited by metabolic inhibitors, dinitrophenol and sodium azide, and by MTX. The transport kinetics at pH 6.0 in the mucosal solution and at pH 7.4 in the serosal solution was saturable with Km of 0.48 +/- 0.23 microM and Vmax of 0.66 +/- 0.24 pmol/cm/min and in addition, the passive diffusion was observed there. These results suggested that the transport of F-MTX was energy-dependent and was mediated by the same transporter as that of MTX, although, in addition to it, other transport mechanism might contribute to the F-MTX transport. Therefore F-MTX will be of great use to investigate the MTX transport system in the normal and diseased states of small intestine, using various fluorescence techniques like visualization of membrane-associated transport proteins.