Transcription factor NF-E2 is crucial for regulating erythroid-specific gene expression. Cloning of the NF-E2 p45 protein has revealed that it contains a basic region-leucine zipper (b-zip) domain which associates with another unidentified protein (of relative molecular mass 18,000) to form functional NF-E2. We show here that products of the maf proto-oncogene family, MafF, MafG and MafK (the small Maf proteins) which possess a b-zip DNA-binding domain but lack a canonical transactivation domain, directly control the DNA-binding properties of p45 by heterodimeric association with p45. Whereas homodimers of the small Maf proteins act as negative regulators, heterodimers composed of Maf and p45 support active transcription in vivo. These results indicate that one (or all) of the small Maf proteins is the second constituent chain required for NF-E2 activity, and that negative as well as positive regulation can be achieved through an NF-E2 site, depending on the equilibrium concentrations of p45 and the Maf proteins inside erythroid cells.