Human brain tissue from nine patients (eight biopsy, one autopsy) was investigated for 3H-phenytoin-binding activity. Protein-related binding was determined by ultrafiltration and was found to resemble experimental animal tissue binding. Phenytoin binding was enhanced by prior lipid removal. Human brain lipid also interacted with 3H-phenytoin as measured by a partition coefficient technique employing hexane. Both human protein and lipid were quantitatively less active than in animals. Measurement of brain, serum, and CSF levels of phenytoin in six patients showed that brain levels were 4 to 10 times higher than free drug as measured in the CSF. It is concluded that under usual clinical circumstances, human brain accumulates phenytoin against a concentration gradient; the accumulative process may be due to binding of phenytoin to tissue proteins and phospholipids.