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Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 ± 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 ± 5.28 ng/106 cells/h) than fraction A cells (73.4 ± 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.