The human CD34 surface antigen is selectively expressed on stem/progenitor cells within the hematopoietic system. Because CD34 expression is tightly linked to the immature status of hematopoietic cells, with expression being rapidly lost as hematopoietic cells mature and differentiate, an examination of its regulation may provide important insights into the molecular control of blood cell development. A comparison of the CD34 nuclear transcription rate in CD34+ and CD34- cells indicated that the CD34 gene is transcriptionally regulated in hematopoietic cell lines. In a previous report, we had identified two major clusters of CD34 transcription initiation sites by 5′ RACE (rapid amplification of cDNA ends) analysis. In transient transfection experiments, we now demonstrate the ability of sequences encompassing each of these clusters to function as promoters of transcription in CD34+ cells. These promoters functioned at equivalent levels in CD34+ and CD34- cells, and the addition of 5′ flanking sequences, extending as far as 3.7 kb upstream, to the core promoters did not differentially modify the level of expression in CD34+ versus the CD34- cell lines. An examination of DNase I hypersensitivity sites within an 18-kb segment of DNA, extending 9 kb either side of the proximal promoter, showed six sites that were primarily associated with CD34- expressing cells. Taken together, these data indicate that the CD34 promoter sequences alone do not confer tissue-or stage-specific expression. Appropriate transcriptional regulation of the CD34 gene must be controlled by chromatin structure, as identified by DNase I hypersensitivity, and/or by other tissue- and stage-specific elements present outside of the promoter region.