CXCL8 is a neutrophil and mast cell chemoattractant that is involved in regulating inflammatory cell influx in asthma. Here, we investigated the transcriptional mechanism involved in CXCL8 induction by TNF-alpha in cultured human airway smooth muscle (HASM) cells and compared these in cells from nonasthmatic and asthmatic individuals. Transfection studies with mutated CXCL8 promoter constructs identified NF-kappaB, activating protein-1, and CAAT/enhancer binding protein (C/EBP)beta as key transcription factors, and binding of these three transcription factors to the CXCL8 promoter after TNF-alpha stimulation was confirmed by chromatin immunoprecipitation analysis. Cells derived from asthmatic individuals produced significantly higher levels of CXCL8 than nonasthmatic cells both basally and following 24 h of stimulation with TNF-alpha (p < 0.001). Furthermore, chromatin immunoprecipitation studies detected increased binding of NF-kappaB p65 and RNA polymerase II to the CXCL8 promoter of asthmatic HASM cells both in the presence and absence of TNF-alpha stimulation. This was not due to either an increased activation or phosphorylation of NF-kappaB per se or to an increase in its translocation to the nucleus. Increased binding of C/EBPbeta to the CXCL8 promoter of unstimulated cells was also detected in the asthmatic HASM cells. Collectively these studies show that HASM cells from asthmatic individuals have increased CXCL8 production due to the presence of a transcription complex on the CXCL8 promoter, which contains NF-kappaB, C/EBPbeta, and RNA polymerase II. This is the first description of an abnormality in transcription factor binding altering chemokine expression in airway structural cells in asthma.