The plasma protein binding of diphenylhydantoin sodium was determined in 26 patients who had been taking diphenylhydantoin regularly for more than two weeks. The free (unbound) diphenylhydantoin level was found to vary from 5.8% to 12.6% of the total drug concentration. This range of variation may be clinically important, if it can be established that the binding of diphenylhydantoin to plasma proteins limits entry of the drug into the brain. The binding of diphenylhydantoin was directly proportional to the plasma albumin concentration, which thus appeared to be the most important determinant of diphenylhydantoin binding. Diphenylhydantoin binding was independent of the plasma concentration of the drug in the usual therapeutic range. In vitro studies showed little effect by phenobarbital sodium or penicillin G; therapeutic levels of acetylsalicylic acid, however, increased free diphenylhydantoin by nearly 50%.