Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the co-existence of Philadelphia-negative with Ph-positive progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. Mononuclear marrow cells from CML patients (n = 20) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers, and allowed to adhere (2 h, 37 degrees C). Following a short-term (3 days) liquid culture, the cells were harvested, incorporated in methylcellulose, and individual colonies were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 9 +/- 20%. The mean (+/- SD) percentages of Ph-negative colonies grown from the stroma-adherent and the stroma-adherent mafosfamide-treated fraction were 41 +/- 32% and 62 +/- 40% (p < or = .005), respectively. Single colony transfer experiments revealed that 50 +/- 13% stroma-adherent and 70 +/- 24% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. In conclusion, the present data demonstrate the possibility to select Ph-negative clones that: 1) have a maintained capability of stroma adherence; 2) are mafosfamide resistant; and 3) have high-replating potential.