PU. 1 (Spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid (granulocytes, monocytes and macrophages) and B cells. PU. 1 is upregulated early during commitment of multipotential progenitors to the myeloid lineages and inhibition of PU. 1 function in human CD34+ progenitors prior to this upregulation blocks myeloid colony formation. Since PU. 1 expression appears to play a role in hematopoietic development, we characterized the PU. 1 promoter. Here we report that the murine PU. 1 promoter, as well as the human promoter, demonstrate tissue-specific reporter gene expression in myeloid cell lines but not in T cells and HeLa (non-hematopoietic cells) cells. Deletion analysis of the PU. 1 promoter indicates that tissue-specific functional elements are encoded in the-61 to-39 bp and-7 to+ 34 bp regions. The first region contains a functional octamer (Oct) site at-54 bp and an Sp1 site at-39 bp. The second contains a binding site at+ 20 bp for both PU. 1 itself and the related ets family member Spi-B. In vivo footprinting assays demonstrate that a hypersensitive band was detected at the PU. 1 site in myeloid cells but not in HeLa. A mutation of the PU. 1 site which abolished PU. 1 binding caused a significant decrease in promoter activity. Mutation of the Oct and/or Sp1 site results in a lesser decrease of promoter activity in myeloid cells. Co-transfection of PU. 1 or Spi-B in cells lacking PU. 1 and Spi-B specifically transactivated a minimal promoter containing the PU. 1 binding site, indicating that PU. 1 can activate its own promoter elements in an autoregulatory loop. Positive autoregulation of the PU. 1 promoter may play an important role in the function of PU. 1 in myeloid cells.