Since in AML differentiation is abnormal but not absent, a hierarchy of stem cells, progenitor cells and more differentiated cells is postulated. The leukemic stem cell might also be characterized by the expression of CD34 and the absence of differentiation markers. Bone marrow samples of 33 AML patients, including 10 patients both at presentation and after relapse, were double labeled for CD34 and CD33. In 14/33 AML less than 1% of the labeled cells were found in the CD34+/33- fraction. After relapse a certain shift towards a more primitive phenotype was observed, but in 4/5 relapsed AML the CD34+/33- fraction remained below 1%. Single cells from the different subfractions were cultured and showed heterogeneous cluster and colony growth in both the CD34-/33+ and CD34+/33+ fraction. More colonies were observed in the CD34+/33- fraction. In AML with a more 'mature' phenotype (low number of CD34+/CD33- cells), highly proliferative myeloid, erythroid and mixed colonies could be cloned exclusively from this small CD34+/33- fraction. In five patients with numerical chromosomal abnormalities all these highly proliferative colonies appeared disomic using in situ hybridization (ISH) with centromeric probes. Based on these data we conclude that the CD34+/33- cell fraction in AML with a more mature immunophenotype (small fraction of cells CD34+/33-) comprise residual normal progenitors, while no primitive leukemic progenitors could be identified.