Deficiency of folic acid, an essential vitamin involved in critical metabolic pathways, occurs in several conditions, including alcoholism. In humans and animal models, chronic ethanol consumption leads to decreased plasma levels and increased urinary levels of folate. An isolated perfused rat kidney model has shown that ethanol produces increased urinary excretion of folate, suggesting a direct effect of ethanol on the kidney. Because the folate binding protein, located in the brush border membrane (BBM) of proximal tubule cells, is thought to be involved in renal folate reabsorption, the effects of ethanol on BBM binding of folate were assessed. Binding studies were conducted using isolated rat kidney cortex BBM preparations, incubated with 3H-labeled 5-methyltetrahydrofolate (5-CH3H4PteGlu) at varying concentrations (0.1-100 nM). Ethanol at 500 mg/dl did not significantly affect [3H]5-CH3H4PteGlu binding in BBM. The structural analogue, folic acid, decreased [3H]5-CH3H4PteGlu binding under similar conditions. Because of the lack of effect of ethanol on binding to isolated BBM, the effects of ethanol probably occur at other steps in the renal uptake and metabolism of folate.