ADAM-TS5 (aggrecanase 2), one of two cartilage aggrecanases is a member of the ADAM protein family. Like ADAM-TS4 (aggrecanase 1) the enzyme cleaves cartilage aggrecan at the Glu³⁷³–Ala³⁷⁴ bond, a marker of aggrecanase activity. In this study we have characterized the substrate specificity of ADAM-TS5 and compared it with that of ADAM-TS4. The recombinant human ADAM-TS5, like ADAM-TS4 cleaves aggrecan at Glu¹⁴⁸⁰–Gly¹⁴⁸¹, Glu¹⁶⁶⁷–Gly¹⁶⁶⁸, Glu¹⁷⁷¹–Ala¹⁷⁷² and Glu¹⁸⁷¹–Leu¹⁸⁷² bonds more readily than at the Glu³⁷³–Ala³⁷⁴ bond. In addition, ADAM-TS5 exhibited an additional site of cleavage in the region spanning residues Gly¹⁴⁸¹ and Glu¹⁶⁶⁷, representing a unique cleavage of ADAM-TS5. ADAM-TS5 cleaved aggrecan approximately 2-fold slower than ADAM-TS4. Neither ADAM-TS5 nor ADAM-TS4 was able to cleave the extracellular matrix proteins fibronectin, thrombospondin, type I collagen, type II collagen, gelatin or general protein substrates such as casein and transferrin. Finally, the zymogen of stromelysin (MMP-3) was not activated by either ADAM-TS4 or ADAM-TS5.