We examined the effect of enteric infusion of 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu), tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu), and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) on concentrations of plasma 5-CH3-H4PteGlu in rats. Concentrations of plasma 5-CH3-H4PteGlu rapidly decreased during continuous bile diversion, whereas initial levels of plasma 5-CH3-H4PteGlu were maintained by means of enteric infusion of 5-CH3-H4PteGlu at a dose of 3 nmol/h, which was approximately equal to total secretion of bile folates. Plasma 5-CH3-H4PteGlu levels were also maintained by the infusion of 5,10-CH2-H4PteGlu, H4PteGlu, or 10-HCO-H4PteGlu at the same dose as 5-CH3-H4PteGlu. The results indicate that folates secreted into the bile are reabsorbed through the intestine to regulate plasma concentrations of 5-CH3-H4PteGlu. We examined the secretion kinetics of bile folates after the intravenous injection of 5-CH3-H4PteGlu or folic acid (PteGlu) at 1 mg/kg body wt. When 5-CH3-H4PteGlu was injected, the bile secretion of folates other than 5-CH3-H4PteGlu increased by < 5 times the base level, whereas that of bile 5-CH3-H4PteGlu markedly increased by approximately 30 times. When PteGlu was given, the bile secretion of nonmethylated tetrahydrofolates markedly increased by 15-27 times, whereas that of 5-CH3-H4PteGlu increased by approximately 7 times. The results suggest that oxidized folates may be one of the sources for bile nonmethylated tetrahydrofolates. It is concluded that nonmethylated tetrahydrofolates in bile play an important role in folate homeostasis through enterohepatic circulation, together with 5-CH3-H4PteGlu.