The aim of this study was to prefabricate an axial bio-synthetic flap for reconstruction of circumferential tracheal defects in a rabbit model. Two series of experiments were performed. In the first set of experiments axial island bio-synthetic flaps were prefabricated. These consisted of an inner island de-epithelialised fasciocutaneous flap from a rabbit's ear and an outer polytetrafluoroethylene vascular graft. The flaps were buried at the base of the rabbit's ear for periods of 1, 2 and 3 weeks (groups A, B and C, respectively), 10 flaps per group. Only one flap in group C failed to survive. Clinical and histological assessment, at the completion of each time period, showed that only the viable flaps of group C developed all the characteristics needed for a tracheal substitute. In the second set of experiments the prefabricated bio-synthetic flaps were transferred to the rabbit's neck by means of microvascular anastomoses. Ten such free flaps were buried at the rabbit's neck for 3 weeks (group D). Eight of the flaps remained viable and all the viable flaps had characteristics similar to those of group C. These results demonstrate the feasibility of creating a prefabricated axial bio-synthetic flap (island or free), over a 3-week period, possessing the characteristics needed for a tracheal substitute in a rabbit model.