To clarify the effect of aspirin on folate balance, we studied serum concentration, protein binding, and urinary excretion of endogenous folate. A healthy woman twice followed an 11-day protocol of constant diet, blood sampling twice daily, collection of all urine, and 650 mg of aspirin by mouth every 4 hours on the middle 3 days. As determined by equilibrium dialysis and Lactobacillus casei assay, aspirin induced a brisk, significant but reversible fall in total and bound serum folate and a small but insignificant rise in urinary folate excretion. Aspirin in vitro also displaced significant amounts of bound serum folate. Thus, aspirin in therapeutic doses can contribute to subnormal serum folate values, and if it increases urinary folate excretion even slightly, may impair folate balance.