Chronic ethanol use can lead to folic acid deficiency in humans. In rats, acute doses of ethanol produce a marked increase in urinary folate excretion, which precedes a decrease in plasma folate levels. To assess the role of ethanol and its metabolism in these effects, five groups of male Sprague-Dawley rats were treated orally as follows: (1) ethanol in 4 doses of 1 g/kg each at 0, 1, 2 and 3 hr; (2) ethanol, as above, plus the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP) at 50 mg/kg IP 15 min prior to 0 hr; (3) glucose in 4 isocaloric doses; (4) glucose plus 4-MP as above; (5) methanol in 4 doses of 1 g/kg. Urinary folate levels (by Lactobacillus casei assay) peaked in both ethanol- and methanol-treated rats at the same time as the urine alcohol levels (6-8 hr) and then declined with a similar time course. Urinary levels of formic acid, which is eliminated by oxidation by a folate-dependent pathway, were significantly increased by ethanol administration, thus indicating another ethanol-folate interaction. Concurrent administration of 4-MP suppressed the increased excretion of formate but had no effect on the increased excretion of folate in ethanol-treated rats. These studies suggest that ethanol has two distinct effects on folate metabolism, one dependent and one independent of ethanol metabolism.