Recently, we have developed a new and fast kinetic method for assessing mitochondrial membrane potential by flow cytometry, based on the quantitation of the initial rate of rhodamine 123 (Rh123) uptake by living cells. This test has proved suitable to detect metabolic and toxic effects on mitochondria. To characterize energy metabolism in a rat hepatoma cell line (N13), we applied this method to assess several metabolic pathways that eventually generate mitochondrial membrane potential. Using this approach, we found that N13 hepatoma cells retain an oxidative capacity comparable with that observed in isolated hepatocytes under the same conditions. These results show that this cell line may represent an adequate biological model to perform metabolic and toxicological studies in vitro.