Tissue Inhibitors Of Metalloproteinase 3 (TIMP3) is an protein belongs to the tissue inhibitor of metalloproteinases family. They are inhibitors of the matrix metalloproteinases. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The activity of recombinant bovine TIMP3 was measured by its ability to inhibit rhMMP2 cleavage of a fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. rhMMP2 was diluted to 100 ug/ml and activated with 1 mM APMA at 37 °C for 1 hour and rbTIMP3 (MW: 23.25 KD) was diluted to different concentrations with the assay buffer. Mix 8 µl of rbTIMP3 curve dilutions, 12.8 µl of activated rhMMP-2, and 59.2 µl of assay buffer, including a control containing assay buffer and the diluted rhMMP-2 and incubate the reactions for 2 hours at 37 °C. Loading 50 µl of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µl of 20 µM substrate. Include a substrate blank containing 50 µl of assay buffer and 50 µl of 20 µM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant bovine TIMP3 significantly decreased rhMMP2 activity. The inhibition IC50 was <15 nM.