This work generated many truncated proteins and Glu 385 to Ala (E 385/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E 385/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH 2-terminal sequence of L 33GRPSEEDEE. A species at 115 kDa and some other protein bands began with F 236VSSHRYV 243, indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R 235–F 236 peptide bond. This cleavage was not an autocatalytic process since the E 385/A mutants were also processed. Furthermore, a 52 kDa band with an NH 2-terminal sequence of L 800KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.