Midkine (MK) is a heparin-binding growth factor, which promotes neurite outgrowth in embryonic neurons and enhances their survival. The three dimensional structure of MK clarified by NMR spectroscopy indicates that several basic amino acids are exposed on the surface of the C-terminal half domain, which retains heparin-binding and neurite-promoting activity. We performed site-directed mutagenesis of these amino acids, and found that mutation of arginine(78) reduced the heparin-binding activity. Mutation of either lysine(88) or lysine(84) scarcely affected heparin-binding activity, while the double mutant involving both lysine residues showed reduction in the activity. Neurite-promoting activity of mutant MKs always correlated with their heparin-binding activity, illustrating the close relationship of the two activities. Thus, the present results verifies the occurrence of two distinct heparin-binding sites involved in neurite-prompting activity of MK molecule. (C) 1997 Academic Press.