Little is known about the long-term properties of fresh cold-stored osteochondral allograft tissue. We hypothesized fresh cold-stored tissue would yield superior material properties in an in vivo ovine model compared to those using freeze-thawed acellular grafts. In addition, we speculated that a long storage time would yield less successful grafts. We created 10-mm defects in medial femoral condyles of 20 sheep. Defects were reconstructed with allograft plugs stored at 4°C for 1, 14, and 42 days; control specimens were freeze-thawed or defect-only. At 52 weeks, animals were euthanized and retrieved grafts were analyzed for cell viability, gross morphology, histologic grade, and biomechanical and biochemical analysis. Explanted cold-stored tissue had superior histologic scores over freeze-thawed and defect-only grafts. Specimens stored for 1 and 42 days had higher equilibrium moduli and proteoglycan content than freeze-thawed specimens. We observed no difference among any of the cold-stored specimens for chondrocyte viability, histology, equilibrium aggregate modulus, proteoglycan content, or hypotonic swelling. Reconstructing cartilage defects with cold-stored allograft resulted in superior histologic and biomechanical properties compared with acellular freeze-thawed specimens; however, storage time did not appear to be a critical factor in the success of the transplanted allograft.