The heparin-binding neurotrophic factor midkine (MK) has been proposed to mediate neuronal cell adhesion and neurite outgrowth promotion by interacting with cell-surface heparan sulfate. We have observed that over-sulfated chondroitin sulfate (CS) D and CS-E show neurite outgrowth-promoting activity in embryonic day (E) 18 rat hippocampal neurons (Nadanaka, S., Clement, A., Masayama, K., Faissner, A., and Sugahara, K. (1998) J. Biol. Chem. 273, 3296-3307). In the present study, various CS isoforms were examined for their ability to inhibit the MK-mediated cell adhesion of cortical neuronal cells in comparison with heparin from porcine intestine and heparan sulfate from bovine kidney. E17-18 rat cortical neuronal cells were cultured on plates coated with recombinant MK in a grid pattern. The cells attached to and extended their neurites along the MK substratum. Cell adhesion was inhibited by squid cartilage over-sulfated CS-E as well as by heparin, but not by heparan sulfate or other CS isoforms. Direct interactions of MK with various glycosaminoglycans were then evaluated using surface plasmon resonance, showing that CS-E bound MK as strongly as heparin, followed by other over-sulfated CS isoforms, CS-H and CS-K. Furthermore, E18 rat brain extracts showed an E disaccharide unit, GlcUAbeta1-3GalNAc(4,6-O-disulfate). These findings indicate that CS chains containing the E unit as well as heparin-like glycosaminoglycans may be involved in the expression and/or modulation of the multiple neuroregulatory functions of MK such as neuronal adhesion and migration and promotion of neurite outgrowth.