CCAAT/enhancer binding protein (C/EBP) ɛ is a recently cloned member of the C/EBP family of transcription factors and is expressed exclusively in cells of hematopoietic origin. The human C/EBPɛ gene is transcribed by two alternative promoters, Pα and Pβ. A combination of differential splicing and alternative use of promoters generates four mRNA isoforms, of 2.6 kb and 1.3–1.5 kb in size. These transcripts can encode three proteins of calculated molecular mass 32.2 kDa, 27.8 kDa, and 14.3 kDa. Accordingly, Western blots with antibodies specific for the DNA-binding domain, that is common to all forms, identify multiple proteins. C/EBPɛ mRNA was greatly induced during in vitro granulocytic differentiation of human primary CD34⁺ cells. Retinoic acid treatment of HL60 promyelocytic leukemia cells for 24 hr induced C/EBPɛ mRNA levels by 4-fold, while prolonged treatment gradually reduced mRNA expression to pretreatment levels. Transient transfection experiments with expression vectors for two of the isoforms demonstrated that the 32.2-kDa protein is an activator of transcription of granulocyte colony-stimulating factor receptor promoter, while the 14.3-kDa protein is not. Thus, C/EBPɛ is regulated in a complex fashion and may play a role in the regulation of genes involved in myeloid differentiation.