The transcription factor PU.1 (Spi-1) is a well-characterized regulator of myeloid and lymphoid development. However, its role in mature functional cells is poorly studied. Here we report the characterization of the novel murine gene pDP4 (PU.1 difference product 4), which is absent from fetal livers of PU.1-deficient mice. pDP4 is transcribed as a single 3.2-kb mRNA with a 1518-base pair open reading frame encoded by 5 exons on chromosome 14. pDP4 expression is strongest in small intestine and bone marrow, in which it is expressed predominately in mature neutrophils. Interestingly, however, pDP4 expression is markedly down-regulated in neutrophils of the peripheral blood and peritoneum. The pDP4 gene encodes a secreted 57-kDa glycoprotein with an olfactomedin-like C-terminus. PU.1 binds to a functional site within the pDP4 promoter, and pDP4 expression in myeloid cells is strictly dependent on PU.1 and the presence of this site. In conclusion, we have identified a novel PU.1-regulated extracellular glycoprotein of the olfactomedin-like family with a possible role in neutrophilic trafficking.