Epithelial　cells　from　tracheal　mucosa　offer　significant　potential　as　a　cell　source　in　development　of　tissue-engineered　trachea.　The　purpose　of　this　study　was　to　investigate　and　optimize　a　suitable　culture　system　for　tracheal　epithelial　cells,　including　the　methods　of　primary　culture,　passage,　identification,　and　cryopreservation.　Epithelial　cells　were　isolated　from　rabbit　tracheal　mucosa　using　tissue　explant　technique　and　were　subjected　to　immunohistochemistry,　immunofluorescence,　and　cryopreservation　after　purification.　Epithelial　cells　reached　confluency　at　14-15　d.　Immunohistochemical　staining　for　cytokeratin　showed　brown　yellow-positive　cytoplasm　and　blue-counterstained　nuclei,　while　immunofluorescence　staining　for　cytokeratin　showed　green-positive　cytoplasm　and　clear　cell　outline,　indicating　that　the　cultured　cells　had　properties　of　epithelial　cells.　After　recovery,　epithelial　cells　exhibited　high　survival　and　viability.　The　results　demonstrated　that　in　vitro　isolation　and　cultivation　model　was　successfully　established　to　provide　high　proliferative　capacity,　typical　morphology　and　characteristics　of　tracheal　epithelial　cells　from　trachea　mucosa　by　the　use　of　the　tissue　explant　technique.