The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at -124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils.