Mouse colon 26 tumor cells were shown to produce collagenase inhibitor in culture. The inhibitor was purified more than 2,000-fold from the culture medium by passage through DE-52 cellulose, CM-52 cellulose, Ultrogel AcA 54, Con A-Sepharose, and Sephadex G-50 Superfine columns. The inhibitor did not bind to Con A-Sepharose as do most other collagenase inhibitors. The inhibitor showed a single band (Mr = 20.5 k) on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and inhibitory activity against interstitial collagenases and gelatinases, except for bacterial collagenase. Double-immunodiffusion analysis using monospecific anti-serum against tissue inhibitor of metalloproteinases (TIMP) from bovine dental pulp showed that colon 26 inhibitor did not cross-react immunologically with the pulp inhibitor. NH2-Terminal protein sequence data were obtained for the first 36 residues of the colon 26 inhibitor, and the first 20 of them exhibited a sequence almost identical with that of a new TIMP recently designated as TIMP-2.