In the bone marrow of C/EBP epsilon(-/-) mice, expression of neutrophil secondary and tertiary granule mRNAs is absent for lactoferrin (LF), neutrophil gelatinase (NG), murine cathelin-like protein (MCLP), and the cathelin B9; it is severely reduced for neutrophil collagenase (NC) and neutrophil gelatinase-associated lipocalin (NGAL). In addition, the expression of eosinophil granule genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBP alpha, C/EBP beta, and C/EBP delta in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo. Stable inducible expression of C/EBP epsilon and C/EBP alpha in the murine fibroblast cell line NIH 3T3 activated expression of mRNAs for B9, MCLP, NC, and NGAL but not for LF. In transient transfections of C/EBP epsilon and C/EBP alpha, B9 was strongly induced with weaker induction of the other genes. C/EBP beta and C/EBP delta proteins weakly induced B9 expression, but C/EBP delta induced NC expression more efficiently than the other C/EBPs. The expression of MBP was inefficiently induced by C/EBP epsilon alone and weakly induced with C/EBP epsilon and GATA-1, but the addition of PU.1 resulted in a striking cooperative induction of MBP in NIH 3T3 cells. Mutation of a predicted PU.1 site in the human MBP promoter-luciferase reporter construct abrogated the response to PU.1. Gel-shift analysis demonstrated binding of PU.1 to this site. MBP and EPX mRNAs were absent in a PU.1-null myeloid cell line established from the embryonic liver of PU.1(-/-) mice. Restitution of PU.1 protein expression restored MBP and EPX protein expression. This study demonstrates that C/EBP epsilon is essential and sufficient for the expression of a particular subset of neutrophil secondary granule genes. Furthermore, it indicates the importance of PU.1 in the cooperative activation of eosinophil granule genes.