Glioblastoma multiforme (glioma) is a malignant lethal neoplasm of the brain which is classified into six subgroups, each based on distinct molecular hallmarks unique to that group. The classical subset of glioma is associated with amplified levels of epidermal growth factor receptor (EGFR), within which distinct, almost exclusively extracellular domain (ECD) mutations can occur. The most common of these, EGFRvIII, has been extensively characterised; however, little remains known about many other potentially important ECD mutations in EGFR in glioma patients. We have selected and studied four novel mutations found in glioma patients residing within Domain IV of EGFR – C260Y, C624F, C628Y and C636Y – which can potentially activate the receptor by forming a dimer via an intermolecular disulphide bond. Expression of these mutants in glioma cells revealed that, surprisingly, they were not more autoactive than wtEGFR. Further analyses showed that only monomeric, fully N-glycosylated mutant receptor attained cell surface expression, with the pre-formed disulfide bonded inactive dimer being underglycosylated and cytoplasmically retained. Interestingly, in response to stimulation by several members of the EGFR ligand family, a conformational shift was induced which facilitated the formation of an active, cell surface disulfide bonded dimer which was derived from the monomeric form of the receptor. The reversible EGFR inhibitors gefitinib (Iressa) and erlotinib (Tarceva) were also able to artificially induce the covalent dimer. Importantly, the inhibitor-induced dimer appeared to be locked into an inactive conformation which was able to incapacitate the ability of EGF to stimulate the receptor, even after inhibitor washout which suggests the dimer is stable after inhibitor removal. This provides evidence that indicates the ECD conformation of the inhibitor-induced dimer may be completely different to that induced by EGF