To confirm the abnormalities of primitive myeloid progenitor cells in patients with severe congenital neutropenia (SCN), we studied their responsiveness to hematopoietic factors including granulocyte colony-stimulating factor (G-CSF). In all SCN patients studied no abnormalities of granulocyte colony-stimulating factor receptor (G-CSFR) gene were detected by polymerase chain reaction-single-strand conformation polymorphism analysis and sequence analysis. A flow cytometric analysis of bone marrow cells based on the expression of CD34, Kit receptor, and G-CSFR demonstrated a reduced frequency of CD34+/Kit+/G-CSFR+ cells in patients with SCN. The granulocyte/macrophage (GM)-colony formation of CD34+/Kit+/G-CSFR+ cells in patients was markedly decreased at all concentrations of G-CSF in serum-deprived semisolid culture. The responsiveness of CD34+/Kit+/G-CSFR+ cells in patients showed a reduced response to the combination of stem cell factor, the ligand for flk2/flt3, and interleukin-3 with or without G-CSF in serum-deprived semisolid and liquid suspension cultures. In contrast, no difference in the responsiveness of CD34+/Kit+/G-CSFR- cells was noted between SCN patients and normal subjects. The bone marrow cells from a patient who underwent bone marrow transplantation showed a restoration of both the reduced frequency and the decreased level of GM-colony formation of CD34+/Kit+/G-CSFR+ cells. These results demonstrate that the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing G-CSFR may play an important role in the impairment of granulopoiesis in patients with SCN.