Matrix metalloproteinase-2 (MMP-2; 72-kDa gelatinase; 72-kDa type IV collagenase; gelatinase A) plays an important role in normal physiological processes and in many pathologic processes such as arthritis and metastasis of cancer. Tissue inhibitor of metalloproteinases-2 (TIMP-2) binds to proMMP-2 or mature MMP-2 at a 1:1 ratio and inhibits the catalytic activity of MMP-2. We demonstrated that the baculovirus/insect cell system does not have TIMP-2 activity. The human proMMP-2 free of TIMP-2 was expressed in the expression system and purified by one-step affinity chromatography using gelatin-Sepharose. The free proMMP-2 was autoactivated to the mature MMP-2 and autodegraded into smaller molecular weight forms in the absence of external activator. The activation and autodegradation of the proMMP-2 was much more rapid in the presence of 4-aminophenylmercuric acetate (APMA). Addition of TIMP-2 inhibits both APMA-induced activation and autodegradation of the free proMMP-2. However, an increasing concentration of TIMP-2 more readily inhibited activation of the free proMMP-2 than autodegradation. These results demonstrate that TIMP-2 plays roles in inhibition of both activation and autodegradation of the free proMMP-2 in addition to inhibition of the catalytic activity of MMP-2.