De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway. In osteosarcoma patients the development of metastases, often to the lungs, is the most frequent cause of death. The aim of this study was to elucidate the molecular mechanisms governing osteosarcoma development and dissemination and, thereby, to identify possible novel drug targets for improved treatment.Osteosarcoma samples were characterized using genome-wide microarrays: increased expression of the EphA2 receptor and its ligand EFNA1 was detected. In addition, increased expression of EFNB1, EFNB3 and EphA3 was suggested. Immunohistochemistry revealed an absence of EphA2 in normal bone, and de novo expression in osteosarcomas. EFNA1 was expressed in normal bone, but was significantly elevated in tumours. Further in vitro investigations on the functional role of EphA2 and EFNA1 showed that EFNA1 ligand binding induced increased tyrosine phosphorylation, receptor degradation and downstream mitogen-activated protein kinase (MAPK) activation. Interference with the MAPK pathway unravelled a potential autoregulatory loop governing mainly EFNA1 expression via the same pathway.Upregulation and de novo expression of ephrins in osteosarcomas are involved in oncogenic signalling and thus might stimulate osteosarcoma metastasis.© 2010 Blackwell Publishing Limited.