Human stem cell factor (hSCF) is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5′ flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5′ end deletions of hSCF gene. This study indicates that the region of - 1190 ∼ -853 significantly enhanced the luc gene expression, while the region of -339 ∼ - 162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both - 1190 ∼ -853 and -339 ∼ -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 ∼ -853 and -339 ∼ -273 DNA fragments of the hSCF 5′ flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.