Glucocorticoids are widely prescribed anti-inflaminatory drugs used for the treatment of many inflammatory lung disorders. However, much still remains unknown about their molecular mechanisms of action. We have previously shown that,glucocorticoid-induced transcription in the lung epithelial cell line NCl-H441 is mediated via C/EBP sites in the promoters of target genes, and is likely to involve the transcription factors C/EBP beta and C/EBP delta. Here, we report that C/EBP beta is the most active C/EBP-factor in both human and mouse lung epithelium and that glucocorticoids induce DNA binding of C/EBP beta in cultured primary mouse lung epithelial cells. Mechanistic studies in H441 cells revealed that glucocorticoids, acting via the glucocorticoid receptor, increase C/EBP beta binding starting 10 min after stimulation. The mechanism is independent of de novo protein synthesis and involves phosphorylation of C/EBP beta at Thr(235). Together this shows that glucocorticoids increase DNA-binding activity of C/EBP beta via posttranslational mechanism(s) involving phosphorylation. (c) 2005 Elsevier Inc. All rights reserved.