The anticonvulsant drug phenytoin was found to inhibit the uptake of folic acid into isolated chick intestinal epithelial cells. At a concentration of 100 micrograms/ml the drug inhibited the cellular accumulation of folic acid by 60%. The efflux of folic acid from preloaded cells was not affected by the drug. The inhibition was observed at an acidic pH of 5.8 and at a neutral pH of 7.4. Both the Na+-dependent and the Na+-independent components of folate uptake were inhibited. Phenytoin had no effect on the uptake of the glucose analog 3-O-methylglucose. Thus it was concluded that the effect of phenytoin on folate uptake was not due to changes in Na+ fluxes, concentration gradients or energy metabolism, and suggested instead a direct effect on the folic acid uptake system.