The binding of diphenylhydantoin-14C (DPH-14C), a CNS depressant, to several purified proteins and a number of tissue fractions from rats and rabbits was studied by ultrafiltration and dynamic dialysis. In all cases the extent of binding correlated very closely with the protein concentration of the fraction investigated. Although binding to purified proteins varied considerably, binding to whole brain, subcellular fractions, liver, heart and kidney were identical. Skeletal muscle bound significantly less than other tissues. When brain homogenates were extracted with acetone or chloroform-methanol (2:1), the insoluble residue bound considerably greater amounts of DPH-14C/mg of protein. Binding decreased after incubation with a proteolytic enzyme, but was not influenced by the addition of cations, EDTA or ouabain. DPH-3H binding was constant over a large range of DPH concentrations (1 .times. 10-10 to 2 .times. 10-4 M) with no evidence for saturable or high affinity binding sites. Tissue protein binding plays a role in the delayed attainment of adequate serum levels and the pharmacologic action of DPH.