Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (Delta TM) and a similar vector coding for the mutation (E240A Delta TM) were constructed for expression in insect cells. Both Delta TM and E240A Delta TM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography, Delta TM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240A Delta TM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.