It has been reported that immunosuppressant-free tracheal transplantation can be achieved by using cryopreserved grafts. However, the ultrastructural changes of cryopreserved tracheal tissue remain unclear. This study investigated this issue in cryopreserved tracheal grafts of 30 Sprague-Dawley (SD) rats, divided into five groups (n = 6 rats/group). Tracheal rings removed from them were stored in a deep freezer at -85 degrees C for 1 week, 1 month, 3 months, or 6 months. Never frozen tracheal tissues were obtained from the control group. Ultrastructural changes in the cryopreserved tracheas of each group were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As the duration of cryopreservation lengthened, tracheal grafts were observed by SEM to develop an obviously speckled depletion of epithelium, accompanied by a loss of cilia in the remaining epithelium. TEM examination of chondrocytes that had been cryopreserved for 3 months revealed obvious injury. Cell membranes and nuclei were frequently observed to be ruptured, and fibrils appeared disorganized and randomly oriented. As the time of cryopreservation increased, ultrastructural injury to tracheal chondrocytes became more severe. The data indicated that the optimal maximum time for cryopreservation of tracheal grafts of SD rats might be <3 months with our methods.