We evaluated the expansion capacity of untreated and 5-fluorouracil (5-FU)-resistant peripheral blood stem cells (PBSC) after 7-day incubation with interleukin-1 (IL-1) plus IL-3 plus stem cell factor (SCF) or with medium alone. We found a significant increase in the proportion of CD34+ cells in the PBSC fraction resistant to 25 micrograms/mL 5-FU after 7-day incubation with IL-1 plus IL-3 plus SCF as compared with the untreated fraction (p = 0.011). We also showed that 5-FU-resistant PBSC have a greater capacity for expansion of IL-1/IL-3/SCF-responsive immature progenitors (p = 0.05), amplification of IL-3 plus GM-CSF responsive progenitors (p = 0.01), and production of committed single growth factor-responsive (granulocyte-macrophage colony-stimulating factor [GM-CSF]) precursors (p = 0.01) than the untreated PBSC. The expansion of all types of progenitors and CD34+ cells was only observed after 7-day incubation with IL-1 plus IL-3 plus SCF. These results suggest that PBSC contain a primitive stem cell population with an enhanced expansion capacity that is identified by 5-FU resistance. As these cells can be expanded in vitro, they may then be suitable for a number of clinical applications.