Abstract Background α5 nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α5 nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α5 nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays. Results Steady expression of α5 nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α5 expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α5 nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α5 is regulated in part by FoxA2, with possible cooperation by GATA-6. Conclusions Our data reveal a highly regulated temporal-spatial pattern of α5 nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α5 in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α5-containing nAChRs.