Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose. Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield. Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated. The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind. The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee [Holmquist, B., & Vallee, B. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6216] that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue. In this case, the hydroxamic acid probably coordinates to the active-site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate. The IC50 for N-(benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes. The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases.