We examined the role of the canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of reduced folate derivatives in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and mutant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hereditarily deficient. In vivo, the biliary excretion of endogenous tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate (5-CH3-H4PteGlu), and 5,10-methylenetetrahydrofolate (5, 10-CH2-H4PteGlu) in EHBR was reduced to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) was detected only in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, significant ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into canalicular membrane vesicles was observed only in SDR. This ATP-dependent uptake was saturable with a Michaelis constant (Km) value of 126 microM, which was comparable with its inhibitor constant (Ki) value of 121 microM for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinitrophenyl-S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the uptake of 5-CH3-H4PteGlu with a Ki of 35 microM, which was similar to its Km value. In addition, H4PteGlu and 5, 10-CH2-H4PteGlu also inhibited the ATP-dependent uptake of DNP-SG. These results indicate that 5-CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefore, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieties.