The retinoic acid (RA)-inducible midkine (MK) gene encodes a heparin-binding protein which can induce neurite outgrowth in cultured mammalian embryonic brain cells. This cytokine shares 65% amino acid sequence identity with another RA-inducible cytokine, pleiotropin (PTN). Both proteins contain 10 conserved cysteine residues, all of which appear to be disulphide linked. MK and PTN are also rich in lysine and arginine residues rendering them susceptible to proteolysis during purification, and making large-scale preparation of these molecules inherently difficult. Recombinant MK has been expressed as a fusion protein using a pGEX vector transfected into E. coli. To enable refolding of MK, the fusion protein was stored in solution at 4 degrees C for 14 days in the presence of dithiothreitol (DTT). Thrombin cleavage of the fusion protein, post storage, typically generated 5 mg of MK per litre of bacterial pellet. To establish the structural integrity of the recombinant product, we have analysed the refolding kinetics and compared the disulphide bond assignment of recombinant MK with that of native MK and native PTN. The synergistic use of micropreparative HPLC, to separate and recover in small eluant volumes enzymatically derived peptide fragments, with matrix assisted laser desorption mass spectrometry (MALD-MS) and N-terminal sequence analysis has allowed the unambiguous identification of the disulphide bonded fragments of native and recombinant MK. The disulphide bond assignment of MK is C12-C36, C20-C45, C27-C49, C59-C91 and C69-C101, and is equivalent to that of PTN.