Using a methodology described by the authors in previous papers, macroporous cellulose membranes possessing large pore sizes (0.4-1 mum) and high porosity (about 55%) were prepared from filter paper by crosslinking or mercerization followed by crosslinking. Epoxy and aniline groups were attached to activate the membrane, and a biomolecule ligand (bovine serum albumin, trypsin or papain) was coupled via diazotization. A detailed comparison between the mercerized and non-mercerized membranes showed that the former was more efficient for biomolecule immobilization. The uniformity of the ligand distribution was investigated using triazine dye and bovine serum albumin as test ligands. The activities of the membranes containing trypsin or papain as ligand for the hydrolysis of N-alpha-benzoyl-L-arginine p-nitroanilide were also determined. Using papain-containing affinity membranes, papain inhibitors were separated from potato tubers.