It was previously shown that patients with chronic myeloid leukemia (CML) have a rare but consistently detectable population of quiescent (G0) leukemic (Philadelphia chromosome-positive and BCR-ABL-positive [BCR-ABL+]) CD34+ cells. In the study described here, most such cells expressed a primitive phenotype (CD38-, CD45RA-, CD71-, and HLA-DR(lo)) and cultures of these cells containing growth factors produced ultimately larger, but initially more slowly growing clones than do cultures of initially cycling CD34+ leukemic cells. Initially quiescent leukemic cells expressing BCR-ABL proliferated in single-cell cultures in the absence of added growth factors, thereby demonstrating their ability to spontaneously exit G0 and enter a continuously cycling state. Interestingly, on isolation, few of these quiescent BCR-ABL+ cells contained either interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF) transcripts, whereas both were present in most cycling BCR-ABL+ CD34+ cells. However, after 4 days of culture in the absence of added growth factors and in association with their entry into the cell cycle (as indicated by up-regulation of Ki-67 and cdc25 transcripts), IL-3 transcripts became detectable. These findings show that entry of leukemic (BCR-ABL-expressing) progenitors into a quiescent (G0) state in vivo is highest among the most primitive leukemic cell populations, associated with a down-regulation of IL-3 and G-CSF gene expression, and spontaneously reversible in association with up-regulation of IL-3 expression. These results highlight the potential physiologic relevance of quiescent CML progenitors, even in treated patients, in whom these cells would be predicted to have a proliferative advantage over their quiescent normal counterparts when cytokine concentrations are low.