Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1α, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1α were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1α subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1α protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1α protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1α transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1α is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1α can be used as a phenotypic marker of nucleus pulposus cells. J. Cell. Biochem. 98: 152–159, 2006. © 2006 Wiley-Liss, Inc.