The aim of this study was to prepare completely autologous heart-valve-shaped constructs without using any artificial scaffold materials by in-body tissue architecture technology, which is a practical concept of regenerative medicine based on the biological defense mechanism against foreign bodies. Silicone rods were used as molds to achieve the tubular shape of the arteries, which were implanted in the subcutaneous spaces of rabbits. After 2 weeks of primary in-body tissue incubation, the silicone rods were completely encapsulated within a thin membranous connective tissue mainly consisting of collagen and having a thickness of approximately 100 microm. To achieve the trileaflet shape of the valve, the cylindrical tissues obtained were rolled up with polyurethane belts cut in the shape of three semi-ovals. The assembled tissues were reimplanted for 2 weeks for secondary incubation. The resulting tissues were over-encapsulated with the newly developed membranous connective tissue having a thickness of approximately 200-400 microm. The newly formed membranes were completely fused to the previously developed inner membrane. After the removal of the two artificial materials, tubular constructs with trileaflet-shaped internal surface were obtained. By controlling the formation of the encapsulating tissue in the two-step in-body tissue incubation process, we were able to develop completely autologous trileaflet valve-shaped constructs.