We have determined the exon organization in the 5' region of the chicken c-mil gene and identified its promoter. A 0.44-kb fragment containing the 5' terminus of the c-mil gene showed strong promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into chicken embryo fibroblasts (CEF). By primer extension analysis, multiple transcriptional start sites were detected within the promoter region. Nucleotide sequence analysis revealed that the c-mil promoter had a high G + C content (71.8%) and contained multiple GC box-like sequences, but no TATA or CAAT boxes. Deletion analysis of 5' upstream sequences showed that the minimal region required for maximal promoter activity in CEF resides in the 99 bp located immediately upstream of the major initiation site. This region contains two putative Sp1 binding sites and one PU box/PEA3 motif, defined as a recognition element for members of the Ets gene family. These sequences bound proteins present in nuclear extracts of CEF as well as in vitro synthesized Ets-related proteins, suggesting that the binding of Sp1 or related proteins and of Ets-related proteins within the promoter is important for modulation of the mil gene.