Abstract Cobalt binding proteins from mouse liver, which were expressed in response to CoCl 2 poisoning, were separated using gel permeation chromatography and then immobilised metal ion affinity chromatography (IMAC) with immobilized cobalt ions. Conditions used in IMAC-Co 2+ were optimised. The fractions eluted with 60 mM imidazole were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). Differences between the samples were also evaluated by a two-dimensional electrophoresis. Samples from the Co 2+-treated mice provided higher number of electrophoretic spots than those from the untreated mice. Relative molecular masses of these proteins are appropriately 37,000; 32,000 and 26,000 and their isoelectric points (p I) are 6.5–7.5.