Human recombinant macrophage-CSF (M-CSF) induced migration across polycarbonate or nitrocellulose filters of human peripheral blood monocytes. Checkerboard analysis of M-CSF-induced migration, performed by seeding different cytokine concentrations above and below the filter, revealed that the locomotory response involved chemotaxis, though some gradient-independent augmentation of migration occurred. Polymixin B did not affect M-CSF chemotaxis and M-CSF was active on monocytes from the LPS-unresponsive mouse strain C3H/HeJ. These findings rule out a contribution of minute endotoxin contamination, below the sensitivity of the Limulus assay, in M-CSF chemotaxis. Rabbit anti-M-CSF antibodies inhibited the chemotactic activity of recombinant M-CSF, thus further indicating that the M-CSF molecule was indeed responsible for chemotaxis. M-CSF preparations encoded by 224 or 522 amino acid cDNA clones were equally effective in inducing monocyte migration. Recombinant M-CSF did not elicit a migratory response in large granular lymphocytes and in endothelial cells under conditions in which appropriate reference attractants were active. A modest stimulation of migration of polymorphonuclear leukocytes, inhibitable by antibodies, was observed at high cytokine concentrations (10 to 100 times higher than those required for monocyte locomotion). The maximal polymorphonuclear leukocytes response evoked by M-CSF was small compared to that evoked by reference chemoattractants or to that evoked by the same cytokine in monocytes. Hence, M-CSF is a potent chemoattractant for mononuclear phagocytes and exerts its action preferentially on cells of the monocyte-macrophage lineage. M-CSF, produced locally by activated macrophages, may play a role in the selective recruitment from the blood compartment of mononuclear phagocytes to amplify resistance against certain noxious agents.