Degradation of the large cartilage proteoglycan aggrecan in arthritis involves an unidentified enzyme aggrecanase, and at least one of the matrix metalloproteinases. Proteinase‐sensitive cleavage sites in the aggrecan interglobular domain (IGD) have been identified for many of the human MMPs, as well as for aggrecanase and other proteinases. The major MMP expressed by chondrocytes stimulated with retinoic acid to degrade their matrix is collagenase‐3 or MMP‐13. Because of its potential role in aggrecan degradation we examined the specificity of MMP‐13 for an aggrecan substrate. The results show that MMP‐13 cleaves aggrecan in the IGD at the same site (..PEN_341‐FFG..) identified for other members of the MMP family, and also at a novel site ..VKP₃₈₄‐VFE.. not previously observed for other proteinases.