Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of α-and β-subunits that belong to the basic helix–loop–helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel α-class hypoxia inducible factor, HIF3α. The HIF3α open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1α and HIF2α (57% and 53% identity, respectively). The C-terminus of HIF3α contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1α. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3α dimerizes with a prototype β-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3α-ARNT interaction can occur in vivo, and that the activity of HIF3α is upregulated in response to cobalt chloride or low oxygen tension.