N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-Lysine (MDP-Lys(L18), muroctasin) is an immunopotentiating substance. Neutrophilia and elevated levels of colony-stimulating factor (CSF) in peripheral blood were previously found after the administration of this compound in both mice and humans. To specify the type of CSF and to elucidate the mechanisms of the neutrophilia, we cultured human peripheral blood mononuclear cells (PBMC) in the presence of muroctasin and measured the levels of granulocyte CSF (G-CSF) in the culture supernatants using our sensitive enzyme-linked immunosorbent assay. G-CSF is an active hematopoietic growth factor specific for cells of a neutrophilic lineage, and muroctasin was found to significantly augment the G-CSF production from PBMC in vitro (P less than 0.01). Furthermore, production of G-CSF from human PBMC in the presence of muroctasin was also supported by the Northern blot analysis using cDNA encoding G-CSF as a probe.