The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH(-) gradient or an inwardly directed H(+)gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.