In previous studies, we found that EDRF2 was an erythroid differentiation related factor, whose expression was markedly upregulated during erythroid differentiation. It suggested that this factor played a role in erythropoiesis. By using rapid amplification of cDNA ends technology, we cloned EDRF2 gene 5 ′- and 3 ′-cDNA ends successfully. Transfection and Northern blot analysis demonstrated that EDRF2 inhibited mRNA expression of a-globin gene, while did not regulate γ-globin gene expression. Gel shift assay confirmed that DNA-binding activity of erythroid specific transcription factors GATA-1, NF-E2 and AP1 was not affected by either forced overexpression or artificial downregulation of EDRF2 gene in K562 cells. However, we detected that the negative regulator of expression of GATA-1 transcription factor was increased in EDRF2 overexpressed K562 cells. These results strongly suggested that EDRF2 was involved in a-globin gene expression and erythroid differentiation and served as a negative regulator of PU.1 transcription factor.