Membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) is a major collagenolytic enzyme that plays a vital role in development and morphogenesis. To elucidate further the structure-function relationship between the human MT1-MMP active site and the influence of the haemopexin domain on catalysis, substrate specificity and inhibition kinetics of the cdMT1-MMP (catalytic domain of MT1-MMP) and the ecto domain ΔTM-MT1-MMP (transmembrane-domain- deleted MT1-MMP) were compared. For substrate 1 [Mca-Pro-Leu-Gly-Leu-Dpa-Ala- Arg-NH2, where Mca stands for (7-methoxycoumarin-4-yl)acetyl- and Dpa for N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl], the activation energy Ea was determined to be 11.2 and 12.2 kcal/mol (1 cal = 4.184 J) for cdMT1-MMP and ΔTM-MT1-MMP respectively, which is consistent with k cat/KM values of 7.37 and 1.46 x 104 M -1·s-1. The kcat/KM values for a series of similar single-stranded peptide substrates were determined and found to correlate with a slope of 0.17 for the two enzyme forms. A triple-helical peptide substrate was predicted to have a kcat/K M of 0.87 x 104 M-1·s-1 for ΔTM-MT1-MMP based on the value for cdMT1-MMP of 5.12 x 104 M-1·s-1; however, the actual value was determined to be 2.5-fold higher, i.e. 2.18 x 104 M-1·s -1. These results suggest that cdMT1-MMP is catalytically more efficient towards small peptide substrates than ΔTM-MT1-MMP and the haemopexin domain of MT1-MMP facilitates the hydrolysis of triple-helical substrates. Diastereomeric inhibitor pairs were utilized to probe further binding similarities at the active site. Ratios of Ki values for the inhibitor pairs were found to correlate between the enzyme forms with a slope of 1.03, suggesting that the haemopexin domain does not significantly modify the enzyme active-site structure.