This study was performed to identify genes that are regulated in the adaptive response to prolonged inhibition of oxidative phosphorylation. Gene microarray analysis in control Clone 9 cells and Clone 9 cells exposed to 5 mM azide for 24 h was carried out as a condition of "Chemical hypoxia." Among several hundred mRNAs whose abundances were either increased or decreased, we noted that the abundance of mRNAs encoding enzymes that catalyze the sequential steps of cholesterol synthesis was decreased; this finding was verified by real-time PCR. Exposure to azide for 24 h markedly inhibited the biosynthesis of cholesterol by approximately 90% and decreased the cellular content of cholesterol by 30%, similar results were observed in HepG2 cells. The abundance of sterol regulatory element binding protein (SREBP)-2 mRNA decreased to 0.37 and 0.25 that of controls after 2 and 24 h exposure, respectively. After 24 h of exposure to azide the precursor and nuclear forms of SREBP-2 protein decreased by approximately 80% and approximately 50%, respectively. Stimulation of AMP-activated protein kinase (AMPK) by AICAR in Clone 9 cells increased the abundance of mRNAs encoding cholesterol biosynthetic enzymes and that of SREBP-1c, and had no effect on SREBP-2 mRNA abundance. We conclude that the decrease in the abundance of multiple mRNAs encoding cholesterol biosynthetic enzymes may be mediated by decreased expression of SREBP-2 mRNA and protein and does not involve stimulation of AMPK. The decrease in SREBP-2 mRNA and protein abundance in the face of decreased cell cholesterol content raises the possibility of a novel regulatory pathway.