Immobilized metal ion affinity chromatography (IMAC) was used to separate various types of recombinant hirudins from the culture broth. The wild type hirudin exhibited a retention in Cu(II)-chelated affinity chromatgoraphy since it contained a single exposed histidine at position 51. To obtain a stronger retention on an IDA-Cu(II) column, the hirudin variants were genetically engineered to contain one or two histidine (s) more than the wild type. While the affinity of the variants for IDA-Cu(II) ligand increased in comparison to that of the wild type, the antithrombin activities reduced to a certain degree. Cu(II), Ni(II) and Zn(II) ions were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. As a result, the Cu(II) chelated chromatography gave the best resolution for all the hirudins tested and appeared to be the only IMAC that could be used generally for the purification of hirudins with a decreasing pH gradient.