AIM: To explore the feasibility of bone tissue regeneration guidance with the frozen heterogeneous periosteum. METHODS: The experiment was conducted in the animal laboratory of Beijing Shijitan Hospital from March to December 2002. ①Preparation of the frozen heterogeneous periosteum: The fresh pigs' cranial or rib bone periosteum was taken and washed with saline solution repeatedly, then defatted by immersion in 0.75 volume fraction alcohol, followed by frozen in the liquor of nitrogen for 10 minutes repeatedly for 3 times. After irradiated under ?-ray up to the amount of 3 kGy, the created membrane was placed in refrigerator for using. ②Surgical procedure: Two artificial mandibular bone defects were performed in 30 adult Japanese white big ear rabbits. One defect was covered with the created membrane as the experimental group, while another as the control group. ③Index observation: The rabbits were sacrificed after 2, 4, 8, 12, 16 weeks postoperatively for macroscopic, radiographic and histopathological observation. RESULTS: All 30 rabbits were involved in the result analysis. ①Macroscopic examination of the two groups: At the eighth week, guiding membrane was observed with irregular border. The experiment group was flat and stiff, while the control group was restored but with soft bone; at the twelfth week, the guiding membrane and surrounding border were unclear, but still could be observed. The newly bone of the experiment group was flat and stiff, while the control group was soft with hollow. ②Histopathological examination: At the eighth week, the new-formed bone in the margin of defect area of the experimental group grew intensively and the inflammatory cells in grafted membrane disappeared; intramembrane fiber was found oedema and broken; at the twelfth week, the trabecular bone of the experimental group was thick and arranged regularly. However, in the control group, the newly formed bone was little in the defect, and the trabecular bone was thinner compared with the experimental group. No evidence of inflammatory reactions was apparent, meanwhile the membrane degraded gradually. ③Radiograph examination: The bone density of the experimental group was greater than the control group at 4 and 8 weeks, and had no significant difference as to the vicinity bone at 12 and 16 weeks; on the other hand, the density of the control group was still lower. CONCLUSION: The frozen heterogeneous periosteum is a useful guiding membrane material for tissue regeneration, because it has no obvious rejection action in body, and could maintain its shape for 8-12 weeks, prevent the fiber tissue from growing into the wound surface, separate different cells and guide tissue regeneration.