We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR-phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated cell-sorting (FACS)-selected 34+DR-and 34+DR+cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR-cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR-cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR-cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC-IC. CML 34+DR+cells and LTC-IC were BCR/ ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 × 105BCR/ABL mRNA-negative 34+DR-cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR-cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation. © 1996 by The American Society of Hematology.