Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by approximately 2.5- and 4-fold, respectively. AA failed to activate c-Jun NH(2)-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-l-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.