The opposing actions of diphenylhydantoin (DPH) and calcium on the level of [32P]phosphate incorporation into particular rat and human brain proteins have been demonstrated in this study by employing the techniques of acrylamide gel electrophoresis and autoradiography. In the presence of calcium several brain proteins showed a marked increase in the incorporation of [32P]phosphate from [gamma-32P]ATP. The calcium-induced increase in phosphorylation of two proteins, designated proteins DPH-L and DPH-M, was significantly inhibited by DPH. DPH inhibited both the calcium-stimulated initial rate and net level of [32P]phosphate incorporated into proteins DPH-L and DPH-M in homogenate and synaptosomal preparations. The data presented in this study are compatible with the hypothesis that the effect of DPH on protein phosphorylation may play an important role in mediating the stabilizing actions of this anticonvulsant on neuronal tissue and seizure discharge.