Overnutrition is one of the major causes of non-alcoholic fattyliver disease (NAFLD). NAFLD is characterized by an accumulationof lipids (triglycerides) in hepatocytes and is often accompaniedby high plasma levels of free fatty acids (FFA). Inthis study, we compared the energy metabolism in acute steatoticand non-steatotic primary mouse hepatocytes. Acute steatosiswas induced by pre-incubation with high concentrationsof oleate and palmitate. Labeling experiments were conductedusing [U-13C5,U-15N2] glutamine. Metabolite concentrationsand mass isotopomer distributions of intracellular metaboliteswere measured and applied for metabolic flux estimation usingtransient 13C metabolic flux analysis. FFAs were efficientlytaken up and almost completely incorporated into triglycerides(TAGs). In spite of high FFA uptake rates and the high synthesisrate of TAGs, central energy metabolism was not significantlychanged in acute steatotic cells. Fatty acid β-oxidationdoes not significantly contribute to the detoxification ofFFAs under the applied conditions.