In the mouse, long-term maintenance of multipotent hematopoietic stem cells in vitro currently requires the establishment of an appropriate adherent layer. When established employing culture medium supplemented with 20% horse serum and 10(-6) M hydrocortisone, the initial adherent layer contains primitive stem cells that are the major contributor of multipotent stem cells (CFUs) assayed subsequently in the supernatant regardless of the addition of fresh bone marrow cells. In contrast, when the adherent layer is established employing 25% fetal calf serum without hydrocortisone, few if any stem cells survive in the adherent layer. Such cultures are dependent upon a recharge with fresh bone marrow as a source of CFUs. A comparison of the latter stem cell-depleted system with the former intact system permits an evaluation of the relative contributions of the adherent layer and stem cells to the long-term maintenance of hematopoiesis in vitro. Studies of the effects of irradiation of the donor animal for the adherent layer and using the intact system demonstrated a reduction in the supernatant CFUs production that was dose- and time-related and evident at doses of 100 and 500 rads. If the adherent layer itself was irradiated immediately before refeeding, a reduction in supernatant CFUs was evident at a dose of 5 rads. These effects, both in vivo and in vitro, cannot be explained solely on the basis of cell killing. Rather, we propose that such doses inactivate, render impotent, or reduce the self-renewal capacity of stem cells that occupy a limited number of "niches" in the adherent layer. Although they are not killed, these impotent stem cells occupy stem cell niches but do not provide an effective contribution of CFUs to the supernatant cells. The adherent layer consists primarily of fibroblasts with significant numbers of macrophages and endothelial cells. The cellular composition of the adherent layer differs between the intact and stem cell-depleted systems in that the latter has a relatively larger proportion of endothelial cells. The composition of the adherent layer influences the type of differentiated cells in the supernatant. The stem cell-depleted adherent layer cultures had a greater proportion of granulocytes among the supernatant cells than macrophages that eventually predominate in the intact system. We have cloned stromal cells from the intact cultures and obtained several stromal cell lines by spontaneous transformation. One of these stromal cell lines (MC1) transfers at least a partial hematopoietic "microenvironment" (granulocytes, macrophages, erythroid cells) to ectopic sites on transplantation to syngeneic recipients.(ABSTRACT TRUNCATED AT 400 WORDS)