The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a “G1-fragment.” To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus …FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus …NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies contribute to the lysis of aggrecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting some diversity in the mode of aggrecan lysis during the excavation of a secondary ossification center. © 2001 Wiley-Liss, Inc.