Self-disintegrating microcapsules were prepared by encapsulating a highly active mix of proteases (Pronase®) into biodegradable polyelectrolyte shells. Pronase was captured by micron-sized calcium carbonate particles that were subsequently embedded into onion-like shells of poly(L-arginine) and poly(L-aspartic acid). EDTA treatment was used to extract the calcium carbonate constituents from the resulting core-shell particles. As a consequence, Pronase was released into the capsule interior and started to digest the surrounding polyelectrolyte shell. Lifetimes of such self-disintegrating capsules could be successfully adjusted to seconds, hours or days by varying the amount of encapsulated Pronase. The enzyme-mediated, sustained release of encapsulated DNA is presented as a prospective application in drug delivery. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA.