An enzymatic procedure for liposome formation through micelle to vesicle transition is described. Amyloglucosidase hydrolysis of dodecyl-beta-D-maltoside (DM) giving dodecyl-P-D-glucoside (DG) leads to dipalmitoylphosphatidylcholine (DPPC)-based vesicle formation from DPPC-DM mixed micelles. Starting from a 1.8 DM/DPPC molar ratio corresponding to mixed micelles, progressive hydrolysis of DM gives DPPC-DG-DM intermediate aggregates ending with DPPC-DG vesicles upon reaction completion. Initial steps of the process corresponding to the exit of the micellar domain were followed by turbidimetry measurements. Next, the reaction progress was investigated by RP-HPLC, HPLC-GEC, and cryofracture electron microscopy. A constant reaction rate is observed in the micellar domain, while the increase of the lamellae proportion considerably decreases the enzyme catalytic activity. Finally, the enzymatic hydrolysis is significally slowed when closed vesicles are formed. Enzymatic activity is dependent on DM availability in the bulk phase and of the DM/DPPC molar ratio in the aggregates, The presence of mixed micelles or lamellar sheets considerably modulates DM monomer concentration in the aqueous phase. The liposomes formed by the enzymatic process are spherical, unilamellar, and heterogeneous in size with a mean diameter ranging from 10 to 80 nm.