Cigarette smoke is a complex mixture of over 4000 constituents. Its effects on cell biology are poorly understood, partly because whole smoke exposure in vitro is technically challenging. To investigate the effects of smoke on cell signalling and function, a 3-D air-liquid interface model of tracheobronchial epithelium, grown from primary human bronchial epithelial cells, was exposed to air or whole mainstream cigarette smoke for 1h in a purpose-designed chamber. Gene expression profiles were then determined at 1, 6 and 24h post exposure using Affymetrix HGU133-2 Plus microarrays. Cells from three different donors were used in the study and the experiment was performed in triplicate for each donor. Genes significantly regulated by smoke, compared to the AIR control, in all experiments were determined. Genes exhibiting differential expression were assigned to functional categories and mapped to signalling pathways. Effects were observed on many cellular processes including xenobiotic metabolism, oxidant/antioxidant balance and DNA damage and repair. Notably, there was marked down-regulation of the transforming growth factor- pathway which has not been previously reported. This study provides important data on the acute effects of whole cigarette smoke on mucociliary epithelium and may be used to gain a greater understanding of smoke toxicity.