The effect of a histidine-excess diet on the hepatic folylpolyglutamate pattern in rat was studied. Rats were fed ad libitum 9.7% casein basal diets with 0.6% methionine (controls) or the basal diets with 3.5% histidine. The average daily weight gain and the food intake in histidine-supplemented rats (His-rats) did not significantly differ from controls. The liver weight in His-rats, however, was 50% higher than controls. Hepatic methyltetrahydropteroylpentaglutamate (CH3-H4PteGlu5), and tetrahydropteroylmonoglutamate concentrations in His-rats was 5.7- and 2-fold higher than controls, respectively. The tetrahydropteroylpentaglutamate (H4PteGlu5) concentration in the His-rats was 74% lower than controls. Considering the homeostasis of folate cofactors in tissues, these results suggest that the hepatic regeneration systems for H4PteGlu5 in His-rats might be repressed and an apparent methylfolate trap might be attained rather on a pteroylpentaglutamate level than a monoglutamate level, and that the activity for catabolizing the excess histidine might exceed the regenerating activity for folate cofactors.