CD34 is currently the only well defined human hematopoietic stem cell marker and is expressed on 1-4% of normal bone marrow cells. Putative binding sites for Ets proteins, a family of transcription factors involved in the regulation of cell differentiation and proliferation in many cell systems, are present in the 5'-flanking region of the CD34 gene. Some of these sites are in close proximity to binding sequences of the encoded product of the proto-oncogene c-myb, which regulates CD34 expression by interacting with the Myb binding sites. Here we demonstrate that Ets-2 (i) transactivates the CD34 promoter in rodent fibroblasts upon interaction with Ets binding sites and (ii) induces expression of CD34 mRNA and protein in the CD34- human glioblastoma T98G cells. Ets-2 and c-Myb transactivate the CD34 promoter independently because specific transactivation is abrogated by site-specific mutations of the binding sites or by competition with oligomers that include wild type but not mutated Myb or Ets binding sites. Ets-2 and c-Myb appear to have addictive effects on transactivation of the CD34 promoter and on induction of CD34 mRNA. Instead, CD34 surface protein levels might be induced synergistically, raising the possibility of a posttranslational mechanism of CD34 expression in cells constitutively expressing c-Myb and Ets-2.