Nanofiltration (NF) membranes were previously used to fractionate peptides in P-lactoglobulin tryptic hydrolysates. It was shown that G-10 NF polyamide membrane coupons with a molecular weight cut-off of 2.5 kg center dot mol(-1) retained all acidic (negatively charged) peptides, making it possible to separate them from basic and neutral peptides. The objective of the work described here was to characterize the ability of G-10 and G-50 NF membranes, which differ by their MWCO, mounted in spiral wound modules to fractionate acid, neutral, and basic peptides at pH 9 and at different peptide concentrations (0.1%, 0.5%, and 1.0%). The selectivity of separation of the peptide was influenced by the Donnan and size exclusion effect for both spiral wound NF membranes. The size exclusion effect was more important with the NF G-50 while Donnan effect was dominant with the NF G-10 membrane. Acidic peptides were completely retained at pH 9 and the transmission of basic and neutral peptides was optimal on the G-10 membrane at the lowest peptide concentration (0.1%).