We have identified a DNA-binding activity with specificity for the βDRE, an evolutionarily conserved transcriptional regulatory element in mammalian adult β-globin promoters. This binding activity, which we term βDRf, for β-globin direct repeat factor, was detected in fractionated nuclear extracts from the murine erythroleukemia cell line and has been partially purified from undifferentiated cells. βDRf makes symmetric contacts on the two copies of its recognition sequence on both strands and introduces a bend into the DNA helix upon binding. While the factor displays a low binding affinity for the βDRE in isolation, it binds to the intact β-globin promoter and DNA fragments containing multiple βDRE-binding sites with high affinity. A correlation between βDRf binding affinity and transcriptional activity of βDRE mutant promoters suggests that this factor stimulates transcription of the β-globin promoter in vivo.