Regulation of the hematopoietic transcription factor PU. 1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU. 1 can lead to leukemia. We previously reported that the PU. 1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU. 1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb− 14 site confers lineage-specific reporter gene expression in vivo. This kb− 14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU. 1 locus between these two conserved regions. DNA binding experiments demonstrated that PU. 1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU. 1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU. 1 gene expression.