Abstract.A specific collagenase has been demonstrated in culture fluids of guinea pig bones. Based on the acrylamide gel eleclrophoretic pattern, the mechanism of action of guinea pig bone collagenase appears similar to other known vertebrate collagenases. Two peaks of enzymatic activity were found upon assay of fractions from gel chromatography, corresponding to molecular weights in the range of 137,000 daltons and 29,000 daltons. The enzyme is inhibited by cysteine and EDTA. Alpha2‐macroglobulin is a much stronger inhibitor of the collagenase than alpha1‐antitrypsin. Activation studies suggested the presence of a proenzyme to guinea pig bone collagenase that was converted to an active enzyme by proteases present in the culture fl