The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) regulates access of 11beta-hydroxyglucocorticoids to the mineralocorticoid receptor by reducing the hydroxyl group of these steroids at position 11. Previous cell culture studies revealed that tumor necrosis factor-alpha (TNF-alpha) down-regulates 11beta-HSD2 activity. Here, we demonstrate that transgenic mice overexpressing TNF-alpha have decreased mRNA abundance and activity of 11beta-HSD2 in kidney tissue, indicating that this effect may occur also in vivo. The analysis of the transcriptional regulation of 11beta-HSD2 by TNF-alpha and phorbol 12-myristate-13-acetate (PMA) with in vivo genomic footprinting in human colon SW620 cells revealed stimulus-dependent protein-DNA interactions in three promoter regions, kappaB1, Sp1/Egr-1I, and Sp1/Egr-1II. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated the relevance of NF-kappaB binding to kappaB1 and of Egr-1 binding to Sp1/Egr-1 sites for the PMA and TNF-alpha effect. We observed a temporal switch of binding to kappaB1 site from active p65/p50 heterodimers to inactive p50/p50 homodimers. TNF-alpha or PMA treatment for 24 h resulted in accumulation of p50 and decrease of p65 nuclear proteins. Overexpression of p50 inhibited HSD11B2 promoter activity and overexpression of Egr-1 inhibited transactivation of the HSD11B2 promoter by p65/p50. In conclusion, TNF-alpha and PMA down-regulate expression and activity of 11beta-HSD2 most likely by a coordinate binding of p50/p50 and Egr-1 to the HSD11B2 promoter.