Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph−) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph− progenitors are highly enriched in the CD34+HLA-DR− fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph− progenitor or stem cells, leaving the unanswered question whether Ph−CD34+HLA-DR- progenitors are normal. To show the clonal nature of Ph−CD34+HLA-DR− CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34+HLA-DR+ and CD34+HLA-DR− progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA−, CD34+HLA-DR−progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34+HLA-DR− derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph−, BCR/ABL mRNA−and polyclonal state in the CD34+HLA-DR−and CD34+HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34+ cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph−, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34+HLA-DR− cells that are Ph− and BCR/ABL mRNA− are polyclonal and therefore benign. This population is suitable for autografting in CML.