The removal of N-terminally located clusters of basic amino acids (N-tail) or C-terminally located clusters of basic amino acids (C-tail) from the midkine (MK) molecule severely reduced its neurite-promoting activity. However, experiments involving chemically synthesized MK derivatives revealed that the roles of the N-tail and C-tail were mostly indirect ones, i.e. they probably maintain the steric arrangements of the N-terminal and C-terminal halves. In particular, the C-domain, which is the C-terminal half devoid of the C-tail, retained considerable neurite-promoting activity when it was uniformly coated on a dish. The removal of the N-tail or C-tail also reduced the enhancing activity of plasminogen activator (PA) in aortic endothelial cells, although the effect was lower. There are two heparin-binding sites in the C-domain, Clusters I and II. A mutation in Cluster I [R78-->Q] affected the PA-enhancing activity only slightly, and a mutation in Cluster II [K83K84-->QQ] abolished the activity, while both mutations are known to reduce the neurite-promoting activity moderately. Therefore, the two heparin-binding sites in the C-domain play different roles in these two activities. Indeed, heparin exhibited different effects on these two activities. We also observed that intact MK was required for ordered neurite-promotion along the path of MK; one possible interpretation of this is that the N-terminal half is necessary for the stability of the molecule. Furthermore, K76 and K99 were found to be required for the secretion of MK; i.e. mutants in which one of these K residues was changed to Q were produced in the host cells, but not found in the medium.