A key challenge in measuring gene expression changes in the lung in response to site-selective toxicants is differentiating between target and nontarget areas. The toxicity for the cytotoxicant 1-nitronaphthalene is highly localized in the airway epithelium. Target cells comprise but a fraction of the total lung cell mass; measurements from whole lung homogenates are not likely to reflect what occurs at the target site. Additionally, the use of generic microarrays to measure expression in airway epithelium may not provide a good representation of transcripts present at the site of toxic action. cDNA libraries from airway and alveolar subcompartments of rat lung were sequenced for the development of a custom microarray representative of these lung regions. We identified 7,460 nonredundant rat lung sequences. Nearly 30% of the sequences on this array are not present on the Affymetrix Rat GeneChip 230. A 20,000-element microarray was developed that delineates differences in gene expression between subcompartments. This is the first in a series of articles employing this microarray for detecting gene expression changes during acute injury produced by 1-nitronaphthalene and subsequent repair.