This work aims to identify of non-reversible structural changes induced in P-lactoglobulin by permeation through porous ultrafiltration membranes. The evaluation of these structural changes is performed using a fluorescence methodology, which combines the use of three different, complementary, fluorescence techniques: steady-state fluorescence, picosecond tirne-resolved fluorescence and steady-state fluorescence anisotropy. The identification of the nature of the structural changes induced upon permeation is possible through comparison of the fluorescence responses obtained for P-lactoglobulin solutions collected after permeation (permeates and retentates) with those induced by chemical (addition of Guanidine hydrochloride, GndHCl) and thermal denaturation of beta-lactoglobulin. The fluorescence approach used allowed to identify irreversible losses of structural integrity of beta-lactogglobulin in the permeates, while beta-lactoglobulin retentates seemed to be unaffected by the ultrafiltration process. The mechanisms that regulate the structural alterations of beta-lactoglobulm and the magnitude of these alterations depend on the protein to membrane pore size ratio, lambda, being more substantial at higher lambda(severe pore constriction). Under these conditions (permeation with a 10 kDa membrane) the structural changes induced in the proteins are dictated by the high shear stress at the membrane pore walls. The increase of the membrane cut-off (30 kDa membrane) induces a decrease in the magnitude of the shear stress and the effect of protein-membrane chemical interactions becomes noticeable. (c) 2007 Elsevier B.V. All rights reserved.