The effects of Bestatin (Ubenimex, UBX) on normal hematopoiesis were investigated using long-term bone marrow cultures (LTBMC) to determine whether it might enhance hematopoiesis over a long period, as well as induce the release of cytokines. LTBMC were inoculated with 0.1 or 1.0 microgram/ml of UBX at the onset of culture and was added at each weekly medium change. The cellularity and the content of the progenitors in nonadherent layer were examined each week for 5 weeks; those from the adherent layer were examined at week 5. The number of the nucleated mature cells and that of the myeloid progenitors from the nonadherent layer increased significantly (approximately two-fold) following treatment with UBX vs controls. The total number of colonies and the number of myeloid progenitors, but not that of erythroid progenitors, from the adherent layer increased significantly (about 1.5-fold) following treatment with 1.0 microgram/ml UBX. The level of interleukin-6 (IL-6) in the culture supernatants was significantly increased one day after the addition of 1.0 microgram/ml UBX. Findings indicate that UBX stimulated both the production of mature cells and myeloid progenitors on normal hematopoiesis in LTBMC. This was mediated by an indirect action via IL-6 production.