Human conducting airways contain two anatomically distinct epithelial cell compartments: surface epithelium and submucosal glands (SMG). Surface epithelial cells interface directly with the environment and function in pathogen detection, fluid and electrolyte transport, and mucus elevation. SMG secrete antimicrobial molecules and most of the airway surface fluid. Despite the unique functional roles of surface epithelia and SMG, little is known about the differences in gene expression and cellular metabolism that orchestrate the specialized functions of these epithelial compartments. To approach this problem, we performed large-scale transcript profiling using epithelial cell samples obtained by laser capture microdissection (LCM) of human bronchus specimens. We found that SMG expressed high levels of many transcripts encoding known or putative innate immune factors, including lactoferrin, zinc alpha-2 glycoprotein, and proline-rich protein 4. By contrast, surface epithelial cells expressed high levels of genes involved in basic nutrient catabolism, xenobiotic clearance, and ciliated structure assembly. Selected confirmation of differentially expressed genes in surface and SMG epithelia demonstrated the predictive power of this approach in identifying genes with localized tissue expression. To characterize metabolic differences between surface epithelial cells and SMG, immunostaining for a mitochondrial marker (isocitrate dehydrogenase) was performed. Because greater staining was observed in the surface compartment, we predict that these cells use significantly more energy than SMG cells. This study illustrates the power of LCM in defining the roles of specific anatomic features in airway biology and may be useful in examining how disease states alter transcriptional programs in the conducting airways.