Gene expression patterns were assessed in normal human bronchial epithelial (NHBE) cells exposed to cigarette smoke condensates (CSC) from commercial cigarettes in order to develop a better understanding of the genomic impact of tobacco exposure, and to define biomarkers that can potentially discriminate tobacco-related effects and outcomes in a clinical setting. NHBE cells were treated with CSCs from two American brands for up to 12 hours in the presence of S9 microsomal fraction from Aroclor 1254-treated rats. High-density oligonucleotide microarrays coupled with a novel statistical analysis that relies on statistical significance levels rather than arbitrary fold-change differences was used to identify genes that undergo expression alterations upon treatment. Expression patterns of approximately 3700 genes were altered after CSC treatments. While a majority of these genes were affected by both CSCs, each condensate also affected a unique subset of approximately 1000 genes. An unexpected finding was that S9, required for metabolizing procarcinogens in CSCs to carcinogenic metabolites, also altered the expression of approximately 1700 genes. Exposure of NHBE cells to different CSCs alters the expression of a large set of genes that affect a common set of biological pathways including those relevant to carcinogenesis. Identification of CSC-affected genes and underlying biological processes may generate an atlas of molecular events that includes biomarkers of tobacco exposure and disease status in smokers. Finally, the finding that S9 affects the expression of a number of genes may have implications for various toxicogenetic assays currently used by regulatory agencies to evaluate harmful effects in exposed humans.