Endochondral bone formation is orchestrated by growth factors produced by chondrocytes and deposited in cartilage matrix. Some of these factors were identified, but their complete list and relationship remains unknown. In this work the growth factors were isolated from calcified cartilage of costochondral junctions. The isolation involved hyaluronidase digestion, guanidinium hydrochloride (GuHCl) extraction, HCl decalcification and GuHCl extraction of decalcified matrix. Growth factors were purified by heparin chromatography and estimated by enzyme-linked immunosorbent assay (ELISA) or used for protein sequence analysis. Bone morphogenetic protein-7 (BMP-7), Growth/differentiation factor 5 (GDF-5), and NEL-like protein 1 (NELL-1) quantitatively dominated in material obtained in all steps of isolation. During ossification perivascular cells and septoclasts enter the cartilage and release growth factors stimulating osteoclastogenesis. Osteoclasts dissolve calcified cartilage and transport released factors needed for stimulation of osteoprogenitor cells. Presence of BMP-7, GDF-5 and NELL-1 at the site of initial bone formation may suggest that their synergistic action favors osteogenesis.