A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPgamma are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPalpha, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPbeta and C/EBPepsilon. However, nuclear C/EBPbeta is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-kappaB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1alpha, and Mip-1beta in this cellular model, whereas TNF-alpha generation was mostly unaffected. Finally, overexpression of a C/EBPbeta mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.