The c-Fes proto-oncogene encodes a myeloid-specific protein-tyrosine kinase that is expressed preferentially in differentiated myeloid cells, but not in early myeloblast progenitor cells. To examine the basis for the phenotypic expression of c-Fes, the transcription initiation sites of the human c-Fes gene were mapped in myeloid leukemia cells and regulatory elements in the genomic c-Fes sequence were characterized. Two major transcription initiation sites were found in the myeloid leukemia cell line THP-1 which delineated exon 1 to be 72-83 bp. When the activity of the CAT reporter gene under the control of the c-Fes promoter region, untranslated exon 1 and intron 1 was measured in TF-1, K562 and MCF-7 cells, only TF-1 cells exhibited chloramphenicol acetyltransferase activity. In contrast, all cell lines supported reporter gene activity when intron 1 was deleted. Deletion analyses revealed a negative regulatory region in intron 1, which was localized by Southwestern analysis and DNA footprinting to a 14 bp region. This negative regulatory region suppressed reporter CAT activity in K562 and TF-1 cells when inserted downstream to the SV40 early promoter. These results suggest that the tissue-specific expression of c-Fes may result, in part, from the negative regulation of transcription in myeloid and nonmyeloid cells.