The transcription factor HIF-1alpha plays a crucial role in modifying gene expression during low oxygen tension. In a previous study, we demonstrated that HIF-1alpha is essential for chondrocyte growth arrest and survival in vivo. To explore further the role of HIF-1alpha in cartilage biology, we undertook studies with primary epiphyseal chondrocytes with a targeted deletion of HIF-1alpha. In this study, we show that HIF-1alpha is necessary for regulating glycolysis under aerobic and anaerobic conditions. HIF-1alpha-null chondrocytes were unable to maintain ATP levels in hypoxic microenvironments, indicating a fundamental requirement for this factor for the regulation of chondrocyte metabolism. Synthesis of the angiogenic factor vascular endothelial growth factor was also significantly induced by hypoxia, and this increase is lost in HIF-1alpha-null mutant cells. Under hypoxic conditions, aggrecan mRNA and protein levels were significantly reduced in chondrocytes lacking the HIF-1alpha transcription factor. Interestingly, strongly increased type-II collagen protein levels were detected in wild-type cells after 44 hours of hypoxia. In addition, type-II collagen mRNA and protein levels were strongly decreased under low oxygen in chondrocytes lacking HIF-1alpha. In summary, our results clearly demonstrate the importance of HIF-1alpha in maintenance of anaerobic glycolysis, and thereby extracellular matrix synthesis, of epiphyseal chondrocytes.