Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma by affinity chromatography on cyanocobalamin-Sepharose and some conventional separation methods. The affinity ligand cyanocobalamin was coupled to AH-Sepharose by a thermolabile linkage. The unsaturated binding protein was absorbed at 4 degrees C and eluted from the column at 37 degrees C as transcobalamin II-cyanocobalamin complex. The final preparation had a specific cyanocobalamin-binding capacity of 0.98 mol cyanocobalamin/mol transcobalamin II, the yield was 55% and the purification index amounted to 1.1 . 10(6). In dodecyl sulphate polyacrylamide gel electrophoresis one major protein band was observed at a molecular weight of 37 000 and a faint band at a molecular weight of 29 000. In polyacrylamide gel isolectric focusing the pure preparation turned out to be heterogeneous with isoelectric points ranging from pH 6.2 to 6.8, possibly by the occurrence of isoproteins.