Background: Cryopreservation of hematopoietic progenitor cells using liquid nitrogen and controlled-rate freezing requires complex equipment and highly trained staff and is expensive. We compared the liquid nitrogen method with methods using a combination of dimethyl sulfoxide (DMSO) and hydroxyethyl starch (HES) for cryopreservation followed by storage in mechanical freezers. STUDY Design and methods: Peripheral blood stem cells (PBSCs) were collected from normal donors by apheresis and allocated to one of four preservation and storage conditions: 1) 10% DMSO with freezing in liquid nitrogen and storage in liquid nitrogen, 2) 5% DMSO and 6% HES with freezing and storage in a -80°C mechanical freezer, 3) 5% DMSO and 6% HES with freezing in a -80°C mechanical freezer and storage in a -135°C mechanical freezer, or 4) 5% DMSO and 6% HES with freezing and storage both in a 135°C mechanical freezer. Cells were stored for 5 years during which total nucleated cells (TNCs), cell viability, CD34+ cell content, and colony-forming unit-granulocyte-macrophage content were determined. Results: There were some significant differences in the variables measured during freezing and the 5 years of storage compared to the values before freezing and storage; however, these differences were not consistent and do not favor one protocol over the others. Samples stored for 24 hours before cryopreservation showed a significant decrease in TNCs, but no other significant changes during the 5 years. Conclusion: In vitro measurements indicate that PBSCs can be successfully frozen and stored using a combination of DMSO and HES providing smaller amounts of DMSO and allowing simplified freezing and storage conditions.