Bone marrow stroma cells from patients with acute myeloid leukemia (AML) display a variety of functional abnormalities. In order to determine whether this is related to an imbalance in the proportion of different stroma cell types or to integration of leukemic progeny into the regulatory cell network, stroma layers were established in mycophenolic acid-treated long-term marrow cultures from 16 patients with AML and 42 controls and analyzed by means of simultaneous membrane immunofluorescence and interphase cytogenetics. Macrophages were identified by CD14 expression, fibroblasts by staining with the AS02 antibody, and malignant cells by leukemia-specific numerical chromosome aberrations, including monosomy 7 and trisomy 8. Compared with normal controls, there was a slight decrease in the proportion of stroma fibroblasts (52+/-27% versus 77+/-5%) in 10-week-old cultures from patients with AML. Two of five AML patients with trisomy 8 and both patients with monosomy 7 had evidence of leukemic stroma cells. Most malignant cells were CD14+ macrophages (3.8-98.1% of all CD14+ cells), but some were AS02+ (2.8-5.2%). AML stroma layers showed a reduced capacity to support the growth of normal hematopoietic cells in standard two-stage long-term cultures, but this was unrelated to the presence or absence of leukemic stroma elements. In conclusion, AML populations vary with respect to their ability to produce a malignant microenvironment. Functional defects in the hematopoietic microenvironment, however, are not limited to AML patients with cytogenetically abnormal stroma cells, but extend to cases without evidence of malignant stroma cells.