Hypoxia plays a key role in the pathophysiology of many disease states, and expression of the retinoic acid receptor-related orphan receptor ␣ (ROR␣) gene increases under hypoxia. We investigated the mechanism for this transient hypoxia-induced increase in ROR␣ expression. Reverse transcription-coupled PCR analysis revealed that the steady-state level of mRNA for the ROR␣4 isoform, but not the ROR␣1 isoform, increased in HepG2 cells after 3 h of hypoxia. Transient transfection studies showed that the hypoxia-induced increase in ROR␣4 mRNA occurs at the transcriptional level and is dependent on a hypoxia-responsive element (HRE) located downstream of the promoter. A dominant-negative mutant of hypoxia-inducible factor-1␣ (HIF-1␣) abrogates the transcription activated by hypoxia as well as the transcription activated by exogenously expressed HIF-1␣, demonstrating the direct involvement of HIF-1␣ in the transcriptional activation. However, HIF-1 alone was not sufficient to activate transcription in hypoxic conditions but, rather, required Sp1/Sp3, which binds to a cluster of GC-rich sequences adjacent to the HRE. Deletion of one or more of these GC boxes reduced or eliminated the HIF-1-dependent transcription. Together, these results suggest that the hypoxia-responsive region of the ROR␣4 promoter is composed of the HRE and GC-rich sequences and that the transcriptional activation under hypoxia is conferred through the cooperation of HIF-1 with Sp1/Sp3.Members of the nuclear receptor superfamily play intrinsic roles in a variety of cellular processes, such as embryogenesis, cell differentiation, cell growth, and cell homeostasis (1, 2). Nuclear receptors, as ligand-dependent transcription factors, activate or repress transcription by binding directly to target genes. They are divided into three groups on the basis of their DNA recognition elements. The first group includes the receptors for steroid hormones, including glucocorticoid and androgen, and recognizes an inverted repeat of an AGAACA half-site motif. The second group, which includes the receptors for nonsteroid hormones such as thyroid hormone, retinoids, and vitamin D, recognizes a direct repeat of an AGGTCA half-site motif. Most of the nuclear receptors so far identified belong to this group. In contrast to these two groups, the third group includes nuclear receptors that recognize and bind to the AG-GTCA half-site motif as a monomer. Little is known about ligands for the members of this group.The ROR 1 subfamily, which has three members (ROR␣, ROR␤, and ROR␥), belongs to the monomeric nuclear receptor subgroup (3). Recently, evidence has been accumulating that ROR␣ is involved in cell differentiation and proliferation. The homozygous mutant mouse staggerer (sg) was initially described as ataxic due to the presence of massive neurodegeneration in the cerebellum. The identification of the widely expressed ROR␣ gene as the site of mutation in the sg mouse has led in recent years to great progress in understanding the molecular basis of its phenotype (4). R...