The receptors for human interleukin-3 (hIL-3R) and granulocyte-macrophage colony-stimulating factor (hGM-CSFR) consist of an x subunit, specific for each cytokine, and a beta subunit, common to IL-3, GM-CSF and IL-5. We cloned genomic DNA covering 1.5 kb of the 5' flanking region of the hIL-3Rx gene and identified multiple transcription start sites by 5'-RACE and primer extension analyses. By use of transient transfection experiments, two regions (nt -363 to -331 and -106 to -92) of the hIL-3Rx promoter appeared to have significant transcription-enhancing activities. Electrophoresis mobility shift assays revealed the binding of Sp1 and unidentified proteins to these regions. Deletion of a putative PU.1 binding site did not affect the promoter activity. We then analyzed 2.5 kb of the hGM-CSFR x gene and found the proximal PU.1 binding site to be important for transcription-enhancing activity, as previously reported. These results suggest that different transcriptional activation mechanisms Lire employed for the transcriptional regulation of hIL-3 and hGM-CSF receptor x genes. (C) 2002 Elsevier Science (USA). All rights reserved.