We describe a novel erythroid cell-specific cDNA (EKLF [erythroid Krüppel-like factor]) isolated by enriching for genes expressed in a mouse erythroleukemia cell line but not expressed in a mouse monocyte-macrophage cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. Additional sequence analyses reveal that the EKLF zinc fingers are most homologous to the Krüppel family of transcription factors and also allow us to predict potential DNA-binding target sites for the EKLF protein. On the basis of this prediction, we show that EKLF is able to bind the sequence CCA CAC CCT, an essential element of the β-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse, and analysis of murine cell lines indicates that EKLF expression is limited to erythroid and mast cell lines. Cotransfection assays establish that EKLF transcriptionally activates a target promoter that contains its DNA-binding site. The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKLF protein may be intimately involved in establishment and/or maintenance of the erythroid cell phenotype.