Objectives: To regenerate of the mastoid air cells and their functions for the treatment of incurable otitis media. Materials and Method: In situ tissue-engineered mastoid air cells using three-dimensional hydroxy apatite (3D-HA) of honeycomb-like structure were used as artificial pneumatic bones. This 3D-HA is made of calcium phosphate and has a high ratio of micropores, 90%. Its surface is coated with collagen. Ten patients (4 males, 6 females) for this clinical study were randomly selected among the patients with severe cholesteatoma, adhesive otitis media, and purulent chronic otitis media, and they were to be received a staged operation. At the first stage of tympanoplasty, collagen-coated 3B-HA was put into the newly opened mastoid cavity, and it was fixed by fibrin glue. Recovery of mastoid aeration and regeneration of the pneumatic air cells of the mastoid cavity were estimated by images of high-resolution computed tomography (HRCT) after the first operation. At the second stage of operation, histopathologic examinations of specimens of HA taken from the reopened mastoid cavity were performed. Results: Aerations in the mastoid cavity were observed in 9 of the 10 patients within 12 months after the second operation. Moreover, the pneumatic structure in the mastoid cavity was partially regenerated in five patients. In these successful cases, mucosa with newly formed capillaries was observed on the surface of the implanted HA at the second stage of operation. In the failure cases, however, connective tissues and granulations invaded the space of the 3D-HA. They were observed as soft-tissue density areas in the HRCT scan images. Conclusions: This study indicates that mucosa can grow on the surface of implanted artificial 3D-HA and can have a gas exchange function in the newly opened mastoid cavity. These tissue-engineered mastoid air cells may be a possible treatment for intractable otitis media.