A sheep animal model was used to investigate the clinical behavior of autologous bone transplants after cryopreservation. The aim of the present study was to compare fresh, cryopreserved and deep-frozen bone transplants in terms of their osseointegration. We used a serum-free cryopreservation protocol with DMSO as cryoprotectant for the bone transplants, which were harvested from the iliac crest of the sheep. The bicortical iliac bone grafts were either cryopreserved or immediately frozen to –80°C for 4 weeks. Four, 8, 12 and 16 weeks after the autologous transplantation of the cryopreserved, fresh or deep-frozen bone transplants to the contralateral iliac crest, the animals were sacrificed and the bone specimens were evaluated clinically, by staining for hematoxylin/eosin and for tartrate-resistant acid phosphatase, by quantified computed tomography, immunohistochemistry (Ki67) and polychrome sequential labeling. The best results were obtained for the fresh specimens with 83% bone healing compared with 75% (cryopreserved bone) and 50% (deep frozen bone). All parameters indicate that bone formation and remodeling processes take place in fresh and cryopreserved transplants. The deep-frozen specimens displayed no fluorochrome uptake in the sequential labeling. These findings indicate that osseointegration of the fresh transplants was the most successful and that osteogenic effects in fresh and cryopreserved transplants are located in the surface area, whereas only the osteoconductive effects are important in the center of the transplants. Thus, cryopreservation is a useful method for the clinical routine because it keeps the osteogenic cells viable, making it superior to deep freezing of abundant bone.