Whole brain concentrations of 3-hydroxybutyrate, glutamate and gamma-aminobutyric acid (GABA) have been measured in two strains of rats with differing susceptibility to obesity. S 5B/Pl rats are resistant to developing obesity when eating a high-fat diet, whereas Osborne-Mendel rats readily develop obesity when eating the same diet. We tested the hypotheses that brain 3-hydroxybutyrate, glutamate and GABA differ between S 5B/Pl rats and Osborne-Mendel rats, and that these substrates/neuroregulators are altered when eating a high-fat diet primarily in S 5B/Pl (resistant) rats. Blood and brain 3-hydroxybutyrate concentrations were higher in S 5B/Pl rats than in Osborne-Mendel rats (p less than 0.05) but diet effects were not significant. Brain glutamate concentration, like 3-hydroxybutyrate, was higher in S 5B/Pl rats than in Osborne-Mendel rats (p less than 0.01) and was not affected by adding fat to the diet. Brain GABA differed only slightly between strains but increased after adding fat to the diet (p less than 0.05) in both strains with a greater increase occurring in S 5B/Pl rats. The brains of S 5B/Pl rats are chronically exposed to higher levels of 3-hydroxybutyrate and glutamate than are those of Osborne-Mendel rats. Thus, 3-hydroxybutyrate is a potential signal in the regulation of body weight. Brain GABA increases with fat feeding, especially in S 5B/Pl rats, suggesting that the ability to adjust to an energy dense diet may be through suppression of food intake by elevated brain GABA.