BACKGROUND The rabbit trachea provides a model system to test the physiological responses of the airways to various agents. Since three-dimensional organization may affect responses of an organ, an organ culture model was established in serum free medium. METHODS Tracheas were fixed in situ, at steps in the preparation of organ cultures, and after one day to three weeks in organ culture. Samples were examined by light microscopy and scanning and transmission electron microscopy for surface morphology, distribution of cell types, and characteristics of the epithelial cell layer. RESULTS The normal tracheal mucosa was discovered to consist of extensive circumferential folds in the cartilaginous portion, which were enhanced upon isolation of the trachea from the animal. The folds consisted principally of differences in epithelial cell height rather than folding of the lamina propria. Enhancement of folding upon removal of the trachea coincided with increased secretion by Clara and possibly mucous cells. In organ culture, epithelial cell height initially increased, producing tall folds and cell types were retained in normal proportions. After prolonged culture, cilia were lost but glandular secretion continued. CONCLUSIONS Changes in the arrangement of basal cells and secretory activity in isolated trachea and in culture may give insight into the functional significance of the epithelial folds.