This article discusses a novel mode of operation for ultrafiltration processes termed Carrier Phase Ultrafiltration (CPUF). CPUF is based on a modification of dead‐end ultrafiltration. Macromolecular fractionation using ultrafiltration is strongly influenced by operating and physicochemical parameters and requires precise “fine‐tuning.” CPUF facilitates the fractionation of high value biological macromolecules at optimised conditions and has several advantages over conventional modes of ultrafiltration. In this article the fractionation of two model proteins, lysozyme (MW 14100) and myoglobin (MW 17000), by CPUF using 25‐kDa MWCO polysulfone membrane is discussed. Fractionation was carried out using two different CPUF modes, 1) pulse input CPUF and 2) step input CPUF. A high recovery of pure product was obtained in each case. These results are compared with those obtained from “conventional” ultrafiltration experiments. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 1–11, 2001.