Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12 degrees C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.