Background: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib (also known as Tarceva or OSI-774) has shown promising response rates in malignant gliomas. We investigated the association between expression of EGFR and downstream signaling components and the response of malignant gliomas to erlotinib in a phase I trial of erlotinib administered either alone or with the alkylating agent temozolomide. Methods: Expression of EGFR and ligand-independent EGFRvIII mutant proteins and of phosphorylated protein kinase B (PKB)/Akt in specimens from glioma patients were assessed by immunohistochemistry. EGFR gene amplification was evaluated by fluorescence in situ hybridization. Mutations in PTEN and EGFR were assessed by polymerase chain reaction amplification and sequencing. Response was evaluated by sequential magnetic resonance imaging every 2 months. The Cochran–Mantel–Haenzel test was used to assess associations between biomarker status and response. All statistical tests were two-sided. Results: Of 41 glioma patients, eight responded to treatment. Response to erlotinib was associated with EGFR expression ( P = .07) and EGFR amplification ( P = .08). These associations were stronger and statistically significant among the 29 patients initially diagnosed with glioblastoma multiforme ( P = .03 and P = .02, respectively). Among six responders with sufficient tumor tissue, none had EGFRvIII mutations. None of the 22 tumors with high levels of phosphorylated PKB/Akt responded to erlotinib treatment, whereas eight of the 18 tumors with low levels of phosphorylated PKB/Akt responded to erlotinib treatment ( P <.001). The level of phosphorylated PKB/Akt was also associated with time to progression ( P <.001). Conclusions: Among glioma patients, those with glioblastoma multiforme tumors who have high levels of EGFR expression and low levels of phosphorylated PKB/Akt had better response to erlotinib treatment than those with low levels of EGFR expression and high levels of phosphorylated PKB/Akt.