Direct interaction between bacteria and epithelial cells may initiate or amplify the airway response through induction of epithelial defense gene expression by nuclear factor-B (NF-B). However, multiple signaling pathways modify NF-B effects in order to modulate gene expression. In this study, the effects of CCAAT/enhancer binding protein (C/EBP) family members on induction of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) was examined in primary cultures of human tracheobronchial epithelial cells incubated with nontypable Haemophilus influenzae. Increased ICAM-1 gene transcription in response to H. influenzae required gene sequences located at -200 to -135 in the 5[[rad]]-flanking region that contain a C/EBP binding sequence immediately upstream of the NF-B enhancer site. Constitutive C/EBP was found to have an important role in epithelial cell ICAM-1 regulation, while the adjacent NF-B sequence binds the RelA/p65 and NF-B1/p50 members of the NF-B family to induce ICAM-1 expression in response to H. influenzae. The expression of C/EBP proteins is not regulated by p38 mitogen-activated protein kinase activation, but p38 affects gene transcription by increasing the binding of TATA binding protein to TATA-box containing gene sequences. Epithelial cell ICAM-1 expression in response to H. influenzae was decreased by expressing dominant-negative protein or RNA interference against C/EBP, confirming its role in ICAM-1 regulation. Although airway epithelial cells express multiple constitutive and inducible C/EBP family member that bind C/EBP sequences, the results indicate C/EBP plays a central role in modulation of NF-B-dependent defense gene expression in human airway epithelial cells after exposure to H. influenzae.