Peripheral blood stem cells (PBSC) contain a mixture of mature and immature hematopoietic progenitors. Resistance to 5-Fluorouracil (5-FU) has been used to identify and characterize primitive quiescent stem cells among bone marrow (BM) cells. To see if the same technique could be used to isolate a similar population of cells among PBSC, low-density peripheral blood mononuclear cells (PBMNC) were collected by cytapheresis in the regenerative phase after high-dose chemotherapy from patients with hematological malignancies. These PBMNC were incubated with increasing concentrations of 5-FU for 24 h. The viable 5-FU resistant cells were then cultured in semi-solid media in the presence of either single cytokines: TCM 5637, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), or a combination of cytokines: interleukin 1 (IL-1) IL-1 + IL-3 + 5637, IL-1 + IL-3 + Stem Cell Factor (SCF). Low concentrations (5-10 micrograms/ml 5-FU) eliminated mature day 7 Colony Forming Units-Granulocyte Macrophage (CFU-GM) and spared day 7 clusters while enriching for day 14 CFU-GM, irrespective of the growth factors used. Higher concentrations of 5-FU (15, 20, 25 micrograms/ml) selected for later forming clonogenic elements. A combination of synergistic growth factors was required for the development of morphologically identifiable clonogenic elements resistant to 25 micrograms/ml 5-FU at day 21 of culture. Further experimentation demonstrated that SCF could effectively replace TCM 5637 in the cytokine combination for the detection of primitive late forming clonogenic elements. The presence of SCF potentiated colony formation by 5-FU resistant PBMNC. It was confirmed that GM-CSF alone was unable to support colony formation by PBMNC resistant to 25 micrograms/ml. These observations demonstrate that PBSC contain a heterogenous mixture of hematopoietic progenitors and that incubation with 25 micrograms/ml 5-FU permits access to a quiescent primitive stem cell population that requires a combination of synergistic growth factors for the development of morphologically identifiable clonogenic elements at day 21. Taken together, these results suggest that PBSC have similar characteristics to BM derived stem cells.