The membrane type 1-matrix metalloproteinase (MTl-MMP) is a membrane-anchored protease that its entire ectodomain is shed from the cell surface. Here we show that in HT1080 cells MTl-MMP is shed as two soluble forms of similar to 52 and similar to 50 kDa. Analyses in purified HT1080 plasma membranes show that release of these species is a two-step time-dependent process that is mediated by integral membrane metalloprotease(s). Differential sensitivity to TIMP-3 inhibition of the shedding process suggests that the second cleavage step leading to the formation of the 50-kDa soluble species is mediated by an ADAM. We also show that shedding of MTl-MMP is independent of its partition into lipid rafts because both wild type and glycosylphosphatidylinositol (GPI)-anchored MTl-MMP are shed. These studies provide new insights into the process of MTl-MMP ectodomain shedding, which may regulate pericellular proteolysis. (c) 2006 Elsevier Inc. All rights reserved.