To develop the preparative method for poly(DL-lactide-co-glycolide)-based microspheres containing proteins, we prepared microspheres from mixture of poly(DL-lactide-co-glycolide) and polydepsipeptide-block-poly(DL-lactide) having cationic or anionic pendant groups. Since the latter amphiphilic copolymers consisting of hydrophobic poly(DL-lactide) segment and hydrophilic polydepsipeptide segment with amino or carboxyl groups could be converted to cationic or anionic block copolymers, they could act as biodegradable surfactants on the preparation of poly(DL-lactide-co-glycolide)-based microspheres by water-in-oil-in-water emulsion method. The amphiphilic block copolymers were established to stabilize primary emulsions on the preparation of microspheres by scanning electron microscopy. We investigated the effects of the addition of the block copolymers on the entrapment efficiency of protein, the release behavior of protein from microspheres and the stability of protein. (C) 2004 Elsevier Ltd. All rights reserved.