Glucocorticoids are widely prescribed anti-inflammatory 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 NCI-H441 is mediated via C/EBP sites in the promoters of target genes, and is likely to involve the transcription factors C/EBPbeta and C/EBPdelta. Here, we report that C/EBPbeta is the most active C/EBP-factor in both human and mouse lung epithelium and that glucocorticoids induce DNA binding of C/EBPbeta in cultured primary mouse lung epithelial cells. Mechanistic studies in H441 cells revealed that glucocorticoids, acting via the glucocorticoid receptor, increase C/EBPbeta binding starting 10 min after stimulation. The mechanism is independent of de novo protein synthesis and involves phosphorylation of C/EBPbeta at Thr(235). Together this shows that glucocorticoids increase DNA-binding activity of C/EBPbeta via post-translational mechanism(s) involving phosphorylation.