The purification of the hydroxynitrile lyase (EC 4.1.2.11, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.