Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. The neutrophils of individuals with SGD display atypical bi-lobed nuclei, lack expression of all secondary and tertiary granule proteins, and possess defects in chemotaxis, disaggregation, receptor up-regulation, and bactericidal activity, resulting in frequent and severe bacterial infections. Previously, a homozygous mutation in theCCAAT/enhancer binding protein–ε (C/EBPε) gene was reported for one case of SGD. To substantiate the role of C/EBPε in the development of SGD and elucidate its mechanism of inheritance, the mutational status of the gene was determined in a second individual. An A-nucleotide insertion in the coding region of the C/EBPε gene was detected. This mutation completely abolished the predicted translation of all C/EBPε isoforms. Microsatellite and nucleotide sequence analyses of the C/EBPε locus in the parents of the proband indicated that the disorder may have resulted from homozygous recessive inheritance of the mutant allele from an ancestor shared by both parents. The mutant C/EBPε₃₂ protein localized in the cytoplasm rather than the nucleus and was unable to activate transcription. Consistent with this, a significant decrease in the levels of the messenger RNAs (mRNAs) encoding the secondary granule protein human 18-kd cationic antimicrobial protein (hCAP-18)/LL-37 and the primary granule protein bactericidal/permeability-increasing protein were observed in the patient. The hCAP-18 mRNA was induced by overexpression of C/EBPε₃₂ in the human myeloid leukemia cell line, U937, supporting the hypothesis that C/EBPε is a key regulator of granule gene synthesis. This study strongly implicates mutation of theC/EBPε gene as the primary genetic defect involved in the development of neutrophil SGD and defines its mechanism of inheritance.