This study was designed to determine whether nutritional folate depletion exerts hepatic oxidative stress in relation to elevated plasma homocysteine. To mimic various extents of folate depletion status in vivo, male Wistar rats were fed an amino acid-defined diet containing either 8 (control), 2, 0.5, or 0 mg folic acid/kg diet. After a 4-wk feeding period, the plasma and hepatic folate concentrations of the rats decreased significantly with each decrement of dietary folate. Folate depletion did not significantly affect two major liver antioxidants: reduced glutathione and alpha-tocopherol. Conversely, folate depletion decreased Cu-Zn superoxide dismutase and glutathione peroxidase activities, but had no effect on catalase activity in liver homogenates. Lipid peroxidation products, as measured by thiobarbituric acid-reactive substances, were significantly higher in livers of folate-depleted rats than in those of the controls. This occurrence of hepatic oxidative stress in folate-depleted rats was confirmed by demonstrating an increased susceptibility of livers of folate-depleted rats to lipid peroxidation induced by additional H2O2 or Fe(2+) treatments compared with the controls. Decreasing dietary folate intake resulted in graded increases in plasma homocysteine concentrations of folate-depleted rats. Elevated plasma homocysteine and decreased plasma and hepatic folate concentrations in folate-depleted rats were all strongly and significantly correlated with increased liver lipid peroxidation (/r/ > or = 0.58, P < 0.0003). These data demonstrate that folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers.