We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma multiforme (GBM), a malignant neoplasm of glial cells, and normal brain. We also initially examined the functional importance of the interaction between EphA2 and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1 in human GBM and normal brain were examined using Western blotting, immunofluorescence, and immunohistochemistry. A functional role for EphA2 was investigated by assessing the activation status of the receptor and the effect of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells. We found EphA2 to be elevated in ∼90% of GBM specimens and cell lines but not in normal brain, whereas ephrinA1 was present at consistently low levels in both GBM and normal brain. EphA2 was activated and phosphorylated by ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent inhibitory effect on the anchorage-independent growth and invasiveness of GBM cells highly overexpressing EphA2, which was not seen in cells expressing low levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM and expressed differentially with respect to its ligand, ephrinA1, which may reflect on the oncogenic processes of malignant glioma cells. EphA2 seems to be functionally important in GBM cells and thus may play an important role in GBM pathogenesis. Hence, EphA2 represents a new marker and novel target for the development of molecular therapeutics against GBM.