BACKGROUND It was previously reported that the combination of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) for 4 days mobilized more primitive CD34+ subsets than did either G-CSF or GM-CSF alone. METHODS The studies determine the optimal number of days of growth factor dosing for mobilization and collection of peripheral blood progenitor cells, by increasing the days of administration of GM-CSF and/or G-CSF or employing the sequential administration of GM-CSF followed by G-CSF. Sixty normal subjects were given injections of G-CSF or GM-CSF alone; GM-CSF and G-CSF concurrently for 4, 5, or 6 days; or a sequential regimen of GM-CSF for 3 or 4 days followed by G-CSF for 2 or 3 days. A 10-L apheresis was performed 24 hours after the last dose. RESULTS The three most efficacious mobilization regimens consisted of sequential GM-CSF for 3 days followed by G-CSF for either 2 or 3 days and G-CSF alone for 5 days. Each of these regimens resulted in the collection of significantly greater numbers of CD34+ cells by apheresis than any of the 4-day dosing regimens with G-CSF and/or GM-CSF (sequential GM-CSF/G-CSF: 3 days/2 days = 3.58 +/- 0.53 x 106 CD34+ cells/kg; GM-CSF/G-CSF: 3 days/3 days = 4.45 +/- 1.08 x 10(6) CD34+ cells/kg; G-CSF: 5 days = 3.58 +/- 0.97 x 10(6) CD34+ cells/kg; all p<0.05 vs. G-CSF and/or GM-CSF for 4 days). Clonogenic assays generally paralleled the level of CD34+ cells. Regimens containing GM-CSF resulted in a higher percentage of the cells from primitive CD34+/CD38-/HLA-DR+ subset than G-CSF alone. CONCLUSIONS Compared with 4-day dosing regimens with G-CSF and/or GM-CSF, mobilization of CD34+ cells in normal subjects using sequential GM-CSF for 3 days followed by G-CSF for 2 or 3 days or using G-CSF alone for 5 days increased the number CD34+ cells that can be collected by a single 10-L apheresis 24 hours after the last dose of cytokine.