Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characters of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH5.5, with smaller Michaelis constant (K_m) for the former (2.4 µM) than for the latter (5.7 µM), indicating its character as a proton-coupled folate transporter which favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characters of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the K_m values were 1.2 µM and 5.8 µM, respectively, for folate and methotrexate, in the rat samll intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1 and we successfully provided several lines of evidences that indicate its role as the molecular entity of the intestinal folate transport system.