The human CD34 hematopoietic stem cell antigen is a highly glycosylated type 1 membrane protein of unknown function. CD34 is expressed on 1% to 4% of bone marrow cells, including pluripotent stem cells and committed progenitors of each hematopoietic lineage. CD34 has also been shown to be expressed on the small vessel endothelium of a variety of tissues and on a subset of bone marrow stromal cells. We have chosen to use the human CD34 gene as model to examine the transcription factors and cis-elements required for stem cell/progenitor cell-specific gene regulation. We show here that the CD34 gene is transcriptionally regulated in tissue culture cells. Using a luciferase reporter gene, we have isolated and characterized an active CD34 promoter. A CD34-luciferase construct, containing 4.5 kb of 5' flanking DNA from a CD34 genomic clone, was 30-fold more active in CD34+ tissue culture cells than in HeLa cells. Sequences from the 3' end of the CD34 gene were shown to have enhancing activity in CD34+ T-lymphoblastic RPMI-8402 cells and not in CD34- U937 cells or in nonhematopoietic HeLa cells. We also show that a cytidine-guanosine island in the 5' end of the CD34 gene is heavily methylated in two CD34- hematopoietic cell lines and demethylated in two CD34+ cell lines. Analysis of the CD34 promoter should result in the identification of stem cell/progenitor cell-specific transcription factors and should provide a means to direct the expression of heterologous genes in hematopoietic stem cells and progenitors.