Phenytoin (PHT) has long been known to cause folate depletion with chronic use. In animal models PHT has been shown to interfere with folate-dependent one-carbon metabolism. Folic acid supplementation in humans has been shown to restore blood levels of folates to normal, but the effects of folic acid supplementation on the PHT-induced effects on one-carbon metabolism have not been addressed. In the present study rats were treated for 8 wk with 1) PHT, 2) folic acid, 3) PHT plus folic acid or 4) vehicle (propylene glycol). Phenytoin treatment caused a decrease in weight gain over the 8 wk of treatment. This effect on weight gain was reversed by folic acid supplementation, but the decrease in brain folate concentration caused by PHT was not reversed by folic acid supplementation, which by itself apparently caused a decrease in brain folate concentration. Phenytoin treatment tended to increase methylation capacity (S-adenosylmethionine:S-adenosylhomocysteine ratio) in the brain and decrease methylation capacity in the liver. Folate supplementation by itself increased methylation capacity in the liver but had no effect in the brain. Folic acid and PHT apparently had independent but opposite effects in the liver, leading to a normalization of methylation capacity. These data suggest that folic acid supplementation in PHT therapy may be effective in reversing the peripheral effects of chronic PHT treatment on one-carbon metabolism but not the central effects.