This study estimates the folate endogenous to a food material (wheat bran) and examines the role of intestinal bacteria in the rat bioassay for folate. After a 4-wk folate depletion period, rats were fed for an additional 4 wk basal diets with or without 0.5% phthalylsulfacetamide and with 100, 200 or 300 g of wheat bran; or 50, 100 or 150 g of xylan; or 0, 0.25, 0.50 or 0.75 mg of folic acid added per kg of basal diet. Xylan increased both liver and fecal folate, and this effect was nearly eliminated by phthalylsulfacetamide. Wheat bran contributed 1.6 micrograms of available folate per g of wheat bran without phthalylsulfacetamide in an apparently valid slope-ratio analysis. With the addition of phthalylsulfacetamide, liver folate increased in rats fed wheat bran diets and decreased in rats fed folic acid diets. The slope-ratio analysis for wheat bran folate with phthalylsulfacetamide became invalid due to a lack of intersection. Phthalylsulfacetamide had no effect on fecal folate excretion from rats fed the wheat bran diets. Further studies are needed on a variety of foods with and without phthalylsulfacetamide to evaluate the effect and importance of intestinal folate synthesis in the rat.