Bestatin, (25, 3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucine, enhanced proliferation of normal human bone marrow granulocyte-macrophage progenitor cells to form CFU-GM colonies in viscous methylcellulose medium in vitro. To elucidate the mechanisms of this effect, the levels of colony-stimulating factor (CSF) in culture supernatant of 1 x 10(6)/ml peripheral blood mononuclear cells (PBMC) cocultured with various concentrations of bestatin were determined using our newly developed sandwich enzyme-linked immunosorbent assay (ELISA). The level of granulocyte/macrophage-CSF (GM-CSF) in the supernatant in the presence of 0.1 microgram/ml bestatin was 312 pg/ml by this ELISA. The production of GM-CSF increased with increasing amounts of added bestatin.