A new ultrafiltration technique based on a multimembrane stack has been developed to fractionate solutes closer in size than conventionally possible. The technique is illustrated here by obtaining a pure protein product from a binary protein mixture. By employing membranes in series without any gaskets or spacers in-between, ultrafiltration is carried out to separate two proteins relatively close in molecular weight or size. Flat YM30 regenerated cellulose membranes, all of the same molecular weight cut-off (MWCO) 30,000, 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 regardless of the physicochemical condition that may be optimal or suboptimal for selective separation by a single membrane. Two systems, myoglobin and β-lactoglobulin, as well as myoglobin and α-lactalbumin were studied, under various operating conditions. The solvent flux reduction encountered when each membrane is added may also be avoided, by operating at increased pressure, while still achieving the desired purification. Cleaning in situ is achievable 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/size. © 2004 Wiley Periodicals, Inc.