Detailed studies were undertaken to better define the role of the liver and the folate enterohepatic cycle in folate homeostasis. Three isotopes of folate were employed in a rat model to study several parameters: (a) intestinal transport; (b) variation in hepatic uptake after different routes of administration; (c) hepatic reduction, methylation, and polyglutamate formation; (d) biliary excretion; (e) transport of folate to tissue and its return to liver for re-entry into the enterohepatic cycle. Folate absorption was not affected by the type of folate administered, but subsequent liver accumulation was greater when PteGlu(1) was given rather than CH(3)H(4)PteGlu(1). After liver uptake, CH(3)H(4)PteGlu(1) is rapidly and quantitatively excreted into bile, whereas nonmethylated folates are either methylated and transported into bile or incorporated into a hepatic polyglutamate pool. Bile folate is then reabsorbed for distribution to both tissue and liver, completing the enterohepatic cycle. The importance of this cycle was demonstrated by long-term bile drainage and by transport studies with two isotopes of CH(3)H(4)PteGlu(1). With bile drainage, serum folate levels fell to 30-40% of normal within 6 h, a much more dramatic drop than that seen with folate-free diets alone. Studies with labeled CH(3)H(4)PteGlu(1) demonstrated that about one-third was taken up by tissue, demethylated, and returned to liver for remethylation and recirculation through the bile and gut. This establishes the enterohepatic cycle as a major factor in folate homeostasis and, for the first time, demonstrates a transport pathway between tissue and liver for nonmethylated folate.