CCAAT/enhancer-binding protein (C/EBP)α and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBPα expressed in 293T cells bound the site centered at –68 most potently. C/EBPα from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPβ did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The –68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the –68 C/EBP-binding site in a −85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPα and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPα-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPα DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPα and C/EBPα-ER with the PU.1 promoter. Finally PU.1 RNA is reduced several-fold in immortalized C/EBPα (−/−) compared with (+/−) cells. Together, these findings indicate that C/EBPα binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPα may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.