The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long-term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8-10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).