Optimal condition for the cryopreservation of canine CD34⁺ cells was explored. Canine bone marrow CD34⁺ cells were isolated from 5 healthy dogs by a magnetic- activated cell-sorting system using a monoclonal antibody specific to canine CD34. These cells were cryopreserved by 4 different methods: 2 different cryoprotectant solutions-solutions A (fetal bovine serum containing 10% dimethylsulfoxide (DMSO) and B (physiological saline containing 5% DMSO, 6% hydroxyethyl starch, and 4% bovine serum albumin)-were used in combination with 2 different freezing procedures-in a rate-controlled programmed freezer (PF) and in an ordinary freezing container. The cell viability, cell recovery rates, and colony-forming unit (CFU) recovery rates were examined following cryopreservation for 1 week, 4 weeks, and 6 months. The values of these parameters were significantly higher for the CD34⁺ cells that had been frozen in Solution B than for those that had been frozen in Solution A, regardless of the freezing procedure employed. The highest CFU recovery rate following cryopreservation for 6 months corresponded to the cells that had been cryopreserved with Solution B and frozen in a PF. In conclusion, cryopreservation with Solution B in a PF proved to be the most efficient of the 4 cryopreservation procedures investigated in terms of maintaining the quality of canine bone marrow-derived CD34⁺ cells. This method will be useful for clinical applications involving the use of canine bone marrow-derived CD34⁺ cells.