Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.