Environment-responsive ultrafiltration membranes were created by modification of a commercial microporous membrane with a hydrogel composed of poly-N-vinyllactams cross-linked with bisacrylamide. The modified membranes were then characterized by their percentage mass gains as well as by their valve effect in response to change in salt concentration in the feed solution. The transmission behavior of model proteins through these membranes was studied using a parameter scanning ultrafiltration technique. A set of membranes selected for highest retention of intermediately sized proteins were examined for protein bioseparation applications. The parameter scanning ultrafiltration technique was then used to examine the feasibility of separating a binary model protein mixture of equine ferritin and human immunoglobulin G by sequential transmission through the environment-responsive membrane. The results obtained demonstrated the potential for using these membranes for multi-component protein separation. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.