Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high- proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet- derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor alpha (TGF-alpha), and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.