Human CD34+ cells purified from frozen mobilized peripheral blood apheresis products (n = 7) were studied immediately (freshly isolated) or refrozen and studied after > 30 days storage in liquid nitrogen (refrozen/thawed). The proliferation and differentiation of freshly isolated or refrozen/thawed CD34+ cells were examined after 10 days of serum-supplemented suspension culture with recombinant human hematopoietic growth factors. The proliferative capacity (fold increase) of the refrozen/thawed CD34+ cells (mean +/- SD, 54.3 +/- 34.3) was comparable to the freshly isolated CD34+ cell cultures (49.0 +/- 42.4). Two-color flow cytometry of the CD34+ cultured cell populations, fresh and refrozen/thawed, displayed typical patterns of neutrophil differentiation into CD15/CD11b neutrophil precursors. The colony-forming ability of freshly isolated and refrozen/thawed CD34+ cells showed no significant differences (p > 0.05) in the total number or type of colony-forming units (CFU-GM, CFU-M, BFU-E, CFU-GEMM) obtained. In addition, the cloning efficiencies of freshly isolated (19.5 +/- 7.6%) and refrozen/thawed CD34+ cells (21.9 +/- 12.7%) were comparable (p = 0.366). These data suggest that CD34+ cells enriched from frozen apheresis blood products can be either used immediately or stored in liquid nitrogen and thawed with minimal effect on their ability to proliferate and differentiate in liquid culture.