A new ultrafiltration technique based on a multimembrane stack has been developed to fractionate proteins closer in molecular weight than conventionally possible. The technique is illustrated here by obtaining a pure protein product from a binary protein mixture. By employing membranes in series using the same membrane without any gaskets or spacers in-between, ultrafiltration is carried out to separate two proteins relatively close in molecular weight. Either flat YM100 regenerated cellulose membranes or Omega 100 K polyethersulfone membranes, of the same molecular weight cutoff (MWCO) 100,000, are stacked together in the desired number, and ultrafiltration takes place. The membrane rejection of a protein is amplified with each additional membrane, ultimately resulting in a completely rejected species. Complete purification of the more permeable protein may be achieved by operating under a physicochemical condition that is optimal for selective separation by a single membrane. The separation of hemoglobin (MW 64,677) and bovine serum albumin (BSA, MW 66,430) was studied under various operating conditions; the molecular weight ratio is 1.03. Complete rejection of bovine serum albumin was achieved using three Omega 100K membranes one on top of the other. To achieve complete rejection in a multimembrane stack, the single membrane rejection must be considerable. Cleaning in situ was achieved with reproducible experimental results before and after on-line cleaning. The results clearly demonstrate that multimembrane stacks can be used for fractionation of proteins that are quite close in molecular weight. (C) 2004 Elsevier B.V. All rights reserved.