Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α gene. Here, we demonstrate that the human GM-CSF receptor α promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor α promoter contains an important functional site between positions 253 and 241 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU. 1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU. 1 site between positions 270 and 254 is involved in positive-negative regulation of the GM-CSF receptor α promoter activity. C/EBPα is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU. 1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor α promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU. 1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor α promoter PU. 1 site. This is the first demonstration of a specific interaction with PU. 1 on a myeloid PU. 1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU. 1 to extracts from certain nonmyeloid cell types which do not express PU. 1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU. 1 sites found on a number of myeloid promoters, and its formation requires an intact PU. 1 site adjacent to a single-stranded region. Expression of PU. 1 in nonmyeloid cells can activate the GM-CSF receptor α promoter. Deletion of the amino-terminal region of PU. 1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor α promoter. Finally, we demonstrate that C/EBPα can also activate the GM-CSF receptor α promoter in nonmyeloid cells. These results suggest that PU. 1 and C/EBPα direct the cell-type-specific expression of GM-CSF receptor α, further establish the role of PU. 1 as a key regulator of hematopoiesis, and point to C/EBPα as an additional important factor in this process.