Bronchiolar stem cells have been functionally defined in vivo on the basis of their resistance to chemical (naphthalene) injury, their infrequent proliferation relative to other progenitor cell types, and their coexpression of the airway and alveolar secretory cell markers Clara cell secretory protein and pro-surfactant protein C, respectively. Cell surface markers that have previously been used for their prospective isolation included Sca-1 and CD34. Using transgenic animal models associated with stem cell expansion, ablation, and lineage tracing, we demonstrate that CD34(pos) cells do not belong to the airway epithelial lineage and that cell surface Sca-1 immunoreactivity does not distinguish between bronchiolar stem and facultative transit-amplifying (Clara) cell populations. Furthermore, we show that high autofluorescence (AF(high)) is a distinguishing characteristic of Clara cells allowing for the fractionation of AF(low) bronchiolar stem cells. On the basis of these data we show that the defining phenotype of the bronchiolar stem cell is CD45(neg) CD31(neg) CD34(neg) Sca-l(low) AF(low). This refinement in the definition of bronchiolar stem cells provides a critical tool by which to assess functional and molecular distinctions between bronchiolar stem cells and the more abundant pool of facultative transit-amplifying (Clara) cells.