OBJECTIVE: Cranioplasty using a cryopreserved skull flap is a wide spread practice. The most well-known complications of cranioplasty are postoperative surgical infections and bone flap resorption. In order to find biological evidence of cryopreserved cranioplasty, we investigated microorganism contamination of cryopreserved skulls and cultured osteoblasts from cryopreserved skulls. METHODS: Cryopreserved skull flaps of expired patients stored in a bone bank were used. Cryopreserved skulls were packaged in a plastic bag and wrapped with cotton cloth twice. After being crushed by a hammer, cancellous bone between the inner and outer table was obtained. The cancellous bone chips were thawed in a water bath of 30°C rapidly. After this, osteoblast culture and general microorganism culture were executed. Osteoblast cultures were done for 3 weeks. Microorganism cultures were done for 72 hours. RESULTS: A total of 47 cryopreserved skull flaps obtained from craniectomy was enrolled. Of the sample, 11 people were women, and the average age of patients was 55.8 years. Twenty four people had traumatic brain injuries, and 23 people had vascular diseases. Among the patients with traumatic brain injuries, two had fracture compound comminuted depressed. The duration of cryopreservation was, on average, 83.2 months (9 to 161 months). No cultured osteoblast was observed. No microorganisms were cultured. CONCLUSION: In this study, neither microorganisms nor osteoblasts were cultured. The biological validity of cryopreserved skulls cranioplasty was considered low. However, the usage of cryopreserved skulls for cranioplasty is worthy of further investigation in the aspect of cost-effectiveness and risk-benefit of post-cranioplasty infection.