Mild abrasion of rat tracheal epithelium results in the death of superficial cells and flattening of basal cells to cover the exposed areas of basal lamina. Six h after injury, colloidal tracers were previously shown to be excluded from the spaces between cells and from the underlying lamina propria. The structural basis for this restoration of barrier function was investigated using transmission electron microscopy of thin sections and freeze fracture replicas. For each of 4 elapsed times after injury, 12 healing lesions in the epithelium were studied extensively. In specimens obtained 2 and 4 h after wounding, apical junctions between epithelium cells could not be identified in either thin sections or freeze fracture replicas. Six h after injury, point contact could be recognized between epithelial cells in thin sections, whereas freezed fracture replicas revealed rudimentary tight junctions consisting of fragmented strands and linearly organized particles. Tweleve h after injury, highly developed tight junctions similar in complexity to those in normal uninjured tracheal epithelium were observed between the relatively undifferentiated basal cells comprising the regenerating epithelium. The focal membrane modification that appears 6 h after injury is an early step in the process of regeneration and restoration of cytologic specialization.