GPI-anchored proteins are localized on the outer layer of plasma membranes and clustered in microdomains generally called lipid rafts. To study the interactions between the lipidic GPI-anchor of the protein and phospholipids, we used phosphatidylcholine monolayers at the air-water interface as a biomimetic membrane system and GPI-alkaline phospliatase prepared from bovine intestinal mucosa (GPI-BIAP) as an GPI-anchored protein model. The monolayer technique allowed us to define GPI-BIAP interaction with DPPC and POPC, lipids differing only by the presence of one unsaturation in their acyl chains. Meanwhile the exclusion pressures were similar for the two phospholipids, the comparison of the Langmuir isotherms (i.e., pressure/area diagrams) indicates that GPI-BIAP interacted differently with DPPC and POPC monolayers. BAM images, acquired in order to visualize the interface organization induced by GPI-BIAP incorporation, confirm these differences. (c) 2005 Elsevier Inc. All rights reserved.