Summary A collagenolytic enzyme capable of degrading reconstituted collagen fibrils at neutral pH and physiological temperature, not demonstrable in the intact forelimb of the adult newt ( Triturus viridescens ) appears in cultures of healing and regenerating tissues of the amputation stump. Tissues proximal to the regenerating region are inactive. The amount of enzyme produced, measured by visible breakdown of thermally reconstituted mammalian collagen gels, and confirmed by release of radioactivty from 14 C-glycine-labeled collagen gels, rises to a peak at 15 days and remains at a high level during the phases of dedifferentiation of the stump and blastema formation, but gradually falls after 20 days as early digital differentiation occurs. Living cells are required for collagenase production since freezing and thawing inactivates the explants.