Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221236 of human cytoskeletal α-actinin. An immunoaffinity purified rabbit polyclonal anti- chicken α-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of α-actinin produced by thermolysin digestion of chicken gizzard α-actinin, but not intact α-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrateresistant acid phosphatase staining. We conclude that an extracellular α-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.