Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP‐1 relative to matrix metalloproteinases (MMPs) promotes progression of fibrosis in liver, lung, and kidney. As to therapeutic strategies, it would be advantageous to antagonize TIMP‐1. MMP‐9 binds TIMP‐1 with high affinity (K_i < 50 pM). Three histidine residues constitute the active site of the enzyme by binding the catalytic zinc. MMP‐9 mutants were generated in which the histidine residues at position 401, 405 or 411, respectively, were replaced by alanine. All mutants were enzymatically inactive as demonstrated by gelatin zymography. In immuno‐precipitation experiments, the MMP‐9 mutant H401A bound 2‐fold more efficiently to TIMP‐1 than H405A, whereas H411A did not bind at all. The mutant H401A was approximately 3‐fold less active in TIMP‐1 binding than wild‐type MMP‐9. Recently, we established cell lines secreting TIMP‐1 constitutively (HepG2‐TIMP‐1 cells). In this cell line, increased expression of TIMP‐1 resulted in suppressed migration and enhanced cell‐cell contact. In co‐cultures of HepG2‐TIMP‐1 cells with HepG2 cells overexpressing the H401A mutant of MMP‐9, the TIMP‐1‐associated phenotype was completely neutralized. These results suggest that a catalytically inactive metalloproteinase (e.g., MMP‐9‐H401A) that still binds TIMP‐1 can be used as a specific antagonist of TIMP‐1 activity in vivo.