A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.
