In order to test the hypothesis that an imbalance between energy requirement and energy supply regulates mitochondrial genes and ultimately mitochondrial biogenesis, energy supply was challenged in HepG2 cells by withdrawal of glucose from the culture medium, making the cells exclusively dependent on mitochondrial ATP production. Such cells showed a 2-fold increase of cytochrome c oxidase activity, elevated levels of mitochondrial DNA, mitochondrial DNA encoded mRNAs and proteins, as well as the nuclear encoded mitochondrial transcription factor A. Lactate production was significantly reduced and glutamine was consumed as an alternative substrate for oxidative metabolism. Long-term adapted cells formed exclusively monolayers, while they normally grow in multilayers forming tumor spheroids. Also, long-term adapted cells proliferated significantly faster. No differences for the ATP/ADP ratio were observed, indicating that this is not the primary signal initiating the adaptative processes. These results show that mitochondrial biogenesis and oxidative metabolism are stimulated in HepG2 cells grown in the absence of fermentable glucose, probably in order to compensate for the diminished supply of glycolytic ATP.