Recent evidence supports a role for thymosin beta 4 (T beta 4) in the inhibition of murine hematopoietic stem cell proliferation. This supposition results from studies in which the N-terminal tetrapeptide derived from native T beta 4 was administered to mice and appeared to prevent CFU-S recruitment into DNA synthesis. The importance of this observation was the concomitant ability of the tetrapeptide to prevent cytosine arabinoside (ara-C) toxicity in mice given LD50 doses of this drug. In the present study, we have extended these observations by demonstrating that whole synthetic T beta 4 is more effective than the N-terminal tetrapeptide in protecting mice from the toxicity of ara-C. This observation supports the hypothesis that T beta 4 is the biologically important parent molecule for this activity. To determine if inhibition of cell cycle progression also occurs in committed human bone marrow progenitors treated with T beta 4, we have investigated the effects of synthetic T beta 4 on proliferating and unstimulated enriched human bone marrow. In short-term liquid cultures studied sequentially over 1-7 days, T beta 4 failed to inhibit cell proliferation, but maintained the proliferative effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on days following maximum stimulation (days 5-7). No effect was noted before the fifth day in culture, nor did T beta 4 exert any demonstrable effect in the absence of added GM-CSF. Any observable effect of T beta 4 required that it be present in the cultures on or before day 3 of GM-CSF stimulation. These results suggest that an additional effect of T beta 4 is the stimulation of a subpopulation of committed human bone marrow precursor cells to become more sensitive to the growth-promoting activity of GM-CSF, thereby enhancing myelopoiesis. It is of interest that the N-terminal peptide of T beta 4 is a shared sequence with tumor necrosis factor alpha, which is also known to have a similar stimulatory capacity. We, therefore, postulate that the growth enhancement noted in short-term cultures is mediated by the region containing these shared sequences.