BACKGROUND: Uncontrolled-rate freezing in -80 ℃ refrigerator is convenient, while controlled-rate freezing in -196 ℃ liquid nitrogen is reliable and long-term, the combination of the two can simplify the process and has been successfully used in clinics. OBJECTIVE: To explore the influences of different cryoprotectants by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen on the cryopreservation of hemopoietic stem cells. METHODS: The experiments were divided into four groups: 10% dimethyl sulfoxide (DMSO) group, 5% DMSO combined with 3% hydroxyethyl starch group, 5% DMSO combined with 0.25 mol/L trehalose group, 5% DMSO combined with 3% hydroxyethyl starch and 0.25 mol/L trehalose group. Peripheral hemopoietic stem cells were cryopreserved by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen. The ultrastructural changes were examined by transmission electron microscopy, the expressions of Annexin-V, PI and Caspase-3 were detected by flow cytometry. RESULTS AND CONCLUSION: There was no significant difference in survival rate, apoptotic rate and necrotic rates of the cryopreserved cells in the four groups (P > 0.05). The ultrastructural changes had no significant difference under the transmission electron microscopy. The viability was more than 90% in frozen-thawed mononuclear cell colonies, and the apoptosis was roughly 50% in the frozen-thawed CD45+ cell population, which contained many mature cells. Of hemopoietic stem cells, early stage cells have greater resistance to damage of cryopreservation than late stage cells. It is concluded that the addition of hydroxyethyl starch or trehalose into DMSO exhibits no synergistic protective effect on the cryopreservation of hemopoietic stem cells.