Normal human bone marrow (BM) contains a small population of cells that can give rise to clonogenic progenitors after 5 weeks in long-term culture (LTC). We have previously shown that these LTC-initiating cells (LTC-IC) differ from the majority of directly clonogenic cells with respect to both light-scattering properties and surface antigen expression. In this paper we show that virtually all LTC-IC (94%) are among the 3%-5% of light-density marrow cells that take up relatively low amounts of rhodamine-123 (Rh-123). In contrast, only 70% of erythroid burst-forming units (BFU-E) and 40% of granulocyte-macrophage colony-forming units (CFU-GM) are recovered in the Rh-123-dull fraction. In addition, we have found that double staining of marrow with Rh-123 and phycoerythrin-labeled anti-CD34 antibodies allows the CD34+ cells to be divided into two subpopulations, of which, on average, 35% are Rh-123-dull. Isolation of these CD34+ Rh-123-dull cells thus provides a single-step enrichment of approximately 240-fold in LTC-IC by comparison to the light-density (less than 1.077 g/cm3) fraction of normal BM. This represents an overall enrichment in LTC-IC of approximately 1000-fold. As expected from the results of staining with Rh-123 only, the majority of directly clonogenic cells are present in the CD34+ Rh-123-bright fraction, where they are enriched approximately 40-fold over their concentration in the light-density fraction. These results indicate marked differences in Rh-123 uptake between subsets of primitive human hematopoietic cells currently defined by different functional assays and suggest that RH-123 staining will be useful for the further purification and analysis of these cells.