Hypoxia increases the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and induces transcription of a variety of genes including vascular endothelial growth factor (VEGF) gene through oxygen sensing mechanisms. In one model, mitochondria-derived reactive oxygen species (ROS) are suggested to be involved in oxygen sensing. Here, we found that hypoxia increased ROS generation and VEGF gene expression in HeLa cells. To further investigate the role of ROS in oxygen sensing, we compared the hypoxic response between rho(0) HeLa cells lacking mitochondrial DNA (EB8) and rho(0) HeLa cells containing mitochondrial DNA from wild-type HeLa cells (HeEB1). The results showed that, although hypoxia markedly increased ROS generation in HeEB1 cells but not in EB8 cells, EB8 cells showed essentially a normal response to hypoxia, as assessed by VEGF gene promoter activity, HIF-1alpha accumulation, and HIF-1 target gene expressions. These results indicate that mitochondria-derived ROS generated in response to hypoxia are not necessary for oxygen sensing in HeLa cells.