Expression of the bactericidal peptide beta-defensin 5 (BNBD5) is strongly induced by bacterial infections of the udder (mastitis). In situ hybridizations showed that bacteria elicit a strong, locally restricted expression of BNBD5 in mammary epithelial cells (MEC). We defined the BNBD5 promoter by primer extension and showed with reporter gene assays in murine HC-11 and primary bovine mammary epithelial cell (pbMEC) cultures that a 1kb segment of the promoter is induced about 3-fold by heat-killed bacteria, LPS, IL-1beta and TNFalpha. Deletion series and point mutations of the promoter showed that NF-IL6 augments the induction, but that NF-kappaB must be bound in cis for pathogen-related stimulation of BNBD5 gene expression. EMSA analyses revealed that both un-stimulated MEC models as well as extracts from healthy udders already display considerable levels of binding competent NF-kappaB. The bacterial stimulus increased this level about 3-fold, as measured with a NF-kappaB driven reporter gene in pbMEC, matching quantitatively the extent of the BNBD5-reporter gene induction. In contrast, expression of the endogenous BNBD5-gene is stimulated much more (>30-fold) in udders and pbMEC indicating that factors other than elevated levels of binding-competent NF-kappaB factors determine the induction of the native gene. Supporting this conclusion, we found that expression of bovine TLR2 or TLR4 in HEK293 cells can reconstitute the bacterial activation of the NF-kappaB expression construct, but not that of the BNBD5-reporter gene. Our data suggest that elevated levels of binding competent NF-kappaB factors mediated via TLR pathogen recognitions mechanisms are not the key switch for pathogen related induction of the BNBD5-encoding gene in MEC.