The myelosuppressive effects of human chemokines were evaluated in vitro on normal myeloid progenitors obtained from bone marrow and cord blood, on bone marrow progenitors from patients with acute or chronic leukemia, on proliferation of human factor-dependent cell line M07e, and in vivo on myelopoiesis in mice. Preincubation of human MIP-1α, MIP-2α, interleukin (IL)-8, platelet factor (PF) 4, monocyte chemotactic and activating factor (MCAF), and interferon-inducible protein-10 (IP-10) in an acetonitrile (ACN) solution significantly enhanced the specific activity of these chemokines for in vitro suppression of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells stimulated to proliferate with a colony stimulating factor plus steel factor (SLF). Combinations of any two of these ACN-treated chemokines synergized to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM at chemokine concentrations below that at which combinations of non-ACN treated chemokines are active. Cord blood progenitors, as previously reported, were in a slow or noncycling state and nonresponsive to inhibition by chemokines. However, after suspension culture with GM-CSF, IL-3, and SLF, they were placed into rapid cell cycle and were responsive to inhibition by ACN-treated chemokines. Low doses of these ACN-pretreated chemokines were active in vivo in suppressing absolute numbers and cycling status of femoral marrow CFU-GM, BFU-E, and CFU-GEMM in C3H/HeJ mice. Other chemokines, alone and in combination, including MIP-1β, MIP-2β, GRO-α NAP-2, and RANTES, were inactive in vitro and in vivo whether or not they were pretreated with ACN. While heterogeneity in responsiveness of CFU-GM from different patients with leukemia to suppression by ACN-treated chemokines was apparent, if the patients had CFU-GM responsive to one of the active chemokines these cells were responsive to the other active chemokines; if patient CFU-GM were not responsive to one of the chemokines, they were not responsive to the other active chemokines. M07e colony-forming cells were responsive to the growth-inhibiting effects of the active ACN-treated chemokines, alone and in combination, but these effects were rapidly reversible and sustained only by multiple daily additions of chemokines. These results should be of value in considering these chemokines for potential clinical use and for assessment of their mechanisms of action, alone and in combination.