In situ hybridization studies allowed for the localization of three MMPs and the angiogenic factor VEGF during secondary ossification. MMPs were widely expressed during ossification of the secondary center, whereas expression of VEGF was restricted to later stages. Introduction: The spatiotemporal expression patterns of the matrix metalloproteinases gelatinase-B (MMP-9), collagenase-3 (MMP-13), and membrane-type 1 metalloproteinase (MMP-14) and the angiogenic peptide vascular endothelial growth factor (VEGF) were studied during development of the proximal epiphysis of the rat tibia. Materials and Methods: Cell expression was analyzed by in situ hybridization. Studies on osteoclastic activity, matrix mineralization, cell proliferation, and vascular progression were also performed. Results: MMP-9, MMP-13, and MMP-14 were expressed in discrete perichondrial cells that gave way to sites of intrachondral canal formation. High expression levels for the three MMPs were found at the blind ends of advancing intrachondral canals and at the expanding borders of the marrow space. Signals for MMP-9 and MMP-13 were in close proximity but did not overlap, whereas MMP-14 was expressed in both MMP-9+ and MMP-13 + cells. VEGF was not expressed during formation of intrachondral vascular canals but was observed in hypertrophic chondrocytes during formation of the bone marrow cavity. Conclusions: Expression of MMPs and VEGF are constant events during development of the secondary ossification center. We propose that MMPs are involved in targeting proteolytic activity during epiphyseal development. VEGF is not expressed during early formation of vascular canals, but it may have a role in the formation of the bone marrow cavity. © 2005 American Society for Bone and Mineral Research.