This study investigates the structural features of the HIF-1α subunit that are essential for heterodimerization, DNA binding, and transactivation. The authors demonstrate that HIF-1α and HIF-1β (ARNT) heterodimerize in the absence of DNA, and in vitro-translated HIF-1α and HIF-1β can reconstitute a HIF-1DNA complex with similar electrophoretic mobility and sequence specificity to endogenous HIF-1. A deletion of the basic domain of HIF-1α eliminates DNA binding without affecting heterodimerization, while a deletion of the carboxy terminus significantly reduces transactivation. Cotransfection assays show that forced expression of recombinant HIF-1α and HIF-1β activates transcription of reporter genes containing EPO enhancer sequences with intact HIF-1 binding sites. Overexpression of a dominant-negative form of HIF-1α blocks reporter gene activation by endogenous HIF-1, further confirming the role of HIF-1 in hypoxia-induced transcriptional activation. These findings provide insights into the molecular mechanisms underlying HIF-1's function in regulating hypoxia-inducible genes.This study investigates the structural features of the HIF-1α subunit that are essential for heterodimerization, DNA binding, and transactivation. The authors demonstrate that HIF-1α and HIF-1β (ARNT) heterodimerize in the absence of DNA, and in vitro-translated HIF-1α and HIF-1β can reconstitute a HIF-1DNA complex with similar electrophoretic mobility and sequence specificity to endogenous HIF-1. A deletion of the basic domain of HIF-1α eliminates DNA binding without affecting heterodimerization, while a deletion of the carboxy terminus significantly reduces transactivation. Cotransfection assays show that forced expression of recombinant HIF-1α and HIF-1β activates transcription of reporter genes containing EPO enhancer sequences with intact HIF-1 binding sites. Overexpression of a dominant-negative form of HIF-1α blocks reporter gene activation by endogenous HIF-1, further confirming the role of HIF-1 in hypoxia-induced transcriptional activation. These findings provide insights into the molecular mechanisms underlying HIF-1's function in regulating hypoxia-inducible genes.