High-resolution protein-protein fractionation is an important and expensive activity in the biotechnological industry. Ultrafiltration (UF), a membrane-based separation process which combines high throughput of product with ease of scale-up is widely used for protein desalting and concentration. In recent years, the potential for achieving high-resolution protein fractionation using ultrafiltration has been demonstrated. However, there has been limited success in translating these into industrial processes. One of the reasons for this is the unsuitability of currently used ultrafiltration process configurations for carrying out high-resolution protein fractionation. These configurations are particularly unsuitable in situations where recovery of pure protein fractions in both permeate and retentate streams is required. This paper discusses a novel three-stage cascade ultrafiltration configuration designed specifically for continuous, high-resolution protein-protein fractionation. The advantages of this configuration over other types are discussed based on results of binary protein fractionation simulation studies. By suitably adjusting the flow streams within this novel configuration it is possible to achieve high recovery as well as high purity of each of the target proteins. The results of simulation show that it is not just the use of the three stages, but how the permeation rates of these three stages are optimized that actually results in high purity and recovery. (C) 2003 Elsevier B.V. All rights reserved.