The development of polyelectrolyte multilayer microcapsules as a delivery system containing bioactive compounds strongly depends on understanding of the major factors that influence capsules loading and release of incorporated substances. Mechanism of protein release from biocompatible polyelectrolyte multilayer microcapsules has been examined using two different approaches of protein encapsulation: (i) "preloading" via coprecipitation of tetramethylrhodamine isothiocyanate (TRITC)-labeled bovine serum albumin (BSA) (TRITC-BSA) into CaCO3 particles followed by multilayer assembly and (ii) "postloading" of TRITC-BSA in preformed empty capsules templated on pure CaCO3 particles taken in the same amount as in "preloading" approach. Polysaccharides (alginate (Alg) or dextran sulfate (Dex)) and polyarginine (PAr) were used as layer constituents. On the basis of the effects of capsule shell composition and thickness, method of protein encapsulation, volume of the surrounding medium, and frequency of medium refreshment on protein release profile, we reveal a mechanism of protein release. The key phenomenon determining the protein release is the property of multilayer polyelectrolyte shells relating to the entrapping and accumulation of protein molecules. The results obtained together with the suggested mechanism of capsule loading and protein release allow us to propose the use of polyelectrolyte microcapsules as a depot system to supply and maintain a defined level of macromolecular drug concentration in surrounding medium.