In this study, a primitive progenitor cell, the blast-cell colony- forming cell (BC-CFC), which is thought by some to be equivalent to the hematopoietic stem cell (HSC), those cells capable of long-term marrow repopulation, has been isolated from normal murine marrow. The cell separation method we employed has, as its final step, the purification of marrow cells based on their ability to take up (bright) or exclude (dull) the mitochondrial dye, Rhodamine (Rho)-123. Rho-bright and Rho- dull cells are enriched for multipotential progenitor cells or for HSC, respectively. It was found that Rho-bright cells contain more BC-CFC than Rho-dull cells (310 +/- 50 v 120 +/- 40 per 10(5) purified cells, respectively). However, the BC-CFC that copurified with the Rho-bright and the Rho-dull cells were different in terms of replating efficiency and response to interleukin-3 (IL-3) and stem cell factor (SCF). In fact, on replating, the blast-cell colonies cultured from the Rho-dull population give rise to many more secondary colonies and had a greater replating efficiency than those obtained from Rho-bright cells (replating efficiency: 29.0 +/- 6.3 v 19.5 +/- 3.7, respectively, P < .01). Furthermore, while the same numbers of blast-cell colonies were detected in culture of Rho-bright cells stimulated with IL-3 alone or in combination with SCF, blast-cell colonies were generated in cultures of Rho-dull cells only in the presence of both IL-3 and SCF. After 5 days in suspension culture stimulated with IL-3 and SCF, Rho-dull cells generated BC-CFC whose replating potential was similar to the replating potential of BC-CFC contained in the Rho-bright population. These results indicate that BC-CFC contained in the Rho-bright and -dull populations are qualitatively different. Because the Rho-dull population contains HSC, the results indicate that few, if any, BC-CFC are HSC.