Ketogenic diets have been used to treat seizure disorders of children and recently it was shown to increase the drug-induced seizure threshold in rats. Protein phosphorylation is a major regulatory mechanism of signal transduction that has been implicated in modulating neuronal excitability. We investigated the basal protein phosphorylation in microslices from different brain regions (hippocampus, cerebral cortex and cerebellum) of young rats fed a ketogenic diet, and we evaluated the effect of this diet on weight development and health of these rats based on serum biochemistry. Thirty-day-old rats consumed ad libitum ketogenic (high fat) or control diets for 8 wk. Rats consuming the high fat diet had ketonemia without signs of undernutrition or illness. Microslices were incubated in media containing ³²P-phosphate, and ³²P-phosphoprotein content was analyzed by one- or two-dimensional electrophoresis followed by autoradiography. Basal protein phosphorylation was greater in brain slices from ketogenic rats. Different increments of synapsin I, GAP-43 and GFAP phosphorylation were observed in two-dimensional autoradiography. A ketogenic diet induced metabolic changes affecting the basal status of protein phosphorylation. This change could affect the mechanisms of signal transduction in neural cells involved in the increase in the seizure threshold.