The aim of this work is to assess the isoelectric separation of a binary protein mixture containing bovine serum albumin (BSA) and lactoferrin (LF) using charged ultrafiltration membranes. First, a thorough characterization of the protein properties that could affect their separation, i.e., the zeta potential under different working conditions, the molecular size, and the tendency to form aggregates, was carried out. The influence of the following operational variables on the separation selectivity was then analyzed: (i) BSA/LF initial concentration ratio, (ii) protein isoelectric point (Ip), and (iii) membrane charge using three composite regenerated cellulose (CRC) membranes: negatively charged, positively charged, and unmodified. The results were used to identify conditions that yielded the optimum separation of proteins. Under those conditions, LF was completely retained in the feed mixture, BSA was characterized by an Ip of pH 5.0, and the use of an unmodified membrane yielded a maximum BSA permeation flux of 30.31 g m(-2) h(-1). By contrast, BSA was completely retained by the negatively charged membrane at an LF Ip of pH 9.0, and LF yielded a maximum permeation flux of 1.07 g m(-2) h(-1). This work provides theoretical and experimental insights into the phenomena that influence the membrane separation of mixtures of similarly sized milk proteins. This work also offers a phenomenological explanation of the main features of the separation process. (C) 2014 Published by Elsevier B.V.