The adherent cell layer in the human long-term marrow culture system provides an in vitro model for the hematopoietic microenvironment. We have devised a two-stage long-term marrow culture system to examine the effect of adherent cell layers from normal and neoplastic cultures on early hematopoietic progenitors. We found that irradiated layers from normal long-term marrow cultures supported granulopoiesis in normal allogeneic marrow cells. This effect, manifested by increased CFU-C production, was amplified when the irradiated layers were overlaid with normal allogeneic marrow initially depleted of these stem cells with cytotoxic anti-Ia (HLA-DR) antibodies. In contrast, there was no regeneration of CFU-C from normal marrow depleted of these progenitors after incubation with irradiated layers from chronic myelogenous leukemia cultures or with passaged marrow fibroblasts. Our data suggest that a functional abnormality may exist in the in vitro microenvironment in chronic myelogenous leukemia and that the two-stage long-term marrow culture system may provide a means of assessing the capacity of adherent cells to support hematopoiesis and an indirect assay for stem cells not measurable with semisolid clonal methods.