Here we investigated encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and between the bilayers. The biological activity (antitrypsin and antichymotrypsin) assay of the protein entrapped in liposomes showed its active sites were spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI confirmed this hypothesis and suggested that this shielding is reversible. The liposomes stability in three various media - artificial gastric juice and intestinal fluids was also examined. The liposomes prepared seem to be promising formulations for BBI and BPTI delivery.