Mammalian globin gene expression is activated through NF-E2 elements recognized by basic-leucine zipper proteins of the AP-1 superfamily. The specificity of NF-E2 DNA binding is determined by several nucleotides adjacent to a core AP-1 motif, comprising a recognition site for transcription factors of the Maf subfamily. Earlier work proposed that p18(MafK) forms a heterodimer with hematopoietic-specific protein p45 NF-E2 to activate transcription through NF-E2 sites. However, there was no direct evidence that p18(MafK) serves this function in vivo; in fact, mice lacking p18(MafK) have no phenotype. Here we describe a novel cDNA clone that encodes the human homolog of chicken MafG. Human MafG heterodimerizes with p45 NF-E2 and binds DNA with specificity identical to that of purified NF-E2 DNA binding activity. A tethered heterodimer of p45 and MafG is fully functional in supporting expression of alpha- and beta-globin, and in promoting erythroid differentiation in CB3, a p45-deficient mouse erythroleukemia cell line. These results indicate that human MafG can serve as a functional partner for p45 NF-E2, and suggest that the p45/MafG heterodimer plays a role in the regulation of erythropoiesis.