The metalloprotease ADAMTS-1, like other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the pro-domain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of the present study were to identify residues of the pro-domain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase which is required for zymogen processing. To gain insight into the putative roles of specific pro-domain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected HEK-293 cells expressing wild-type (wt) and pro-domain mutants of proADAMTS-1. Cells expressing wtADAMTS-1 initially produced a 110 kDa zymogen form that was later converted to an 87 kDa form which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87 kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 pro-domain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent BFA and monensin suggest that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the pro-domain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with Alanine of selected residues Cys106, Tyr108, Gly110, Cys125, Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the pro-domain of ADAMTS-1 are involved in its biosynthesis.