Human malignant glioma cell lines and adult brain tumors overexpress high levels of interleukin-13 receptor alpha2 chain (IL-13Ralpha2). Because the IL-13Ralpha2 chain is an important target for cancer therapy and prognosis for patients with brainstem glioma (BSG) remains dismal, we investigated the expression of this receptor in specimens of diffusely infiltrative pediatric BSG relative to normal brain tissue. Twenty-eight BSG specimens and 15 normal brain specimens were investigated for IL-13Ralpha2 protein expression by immunohistochemical analysis (IHC) using two different antibodies in two different laboratories. Highly sensitive Q-dot-based IHC and in situ hybridization (ISH) assays were also developed to identify IL-13Ralpha2 protein and RNA in these specimens. The results were evaluated independently in two laboratories in a blinded fashion. By Q-dot IHC or a standard IHC assay, 17 of 28 (61%) tumor specimens showed modest to strong staining for IL-13Ralpha2, while 15 normal brain tissue samples showed weak expression for IL-13Ralpha2 protein. Significant interrater agreement between the two laboratories was seen in the assessment of IL-13Ralpha2 intensity. High-level IL-13Ralpha2 RNA expression was detected in tumor samples by Q-dot ISH, but only weak RNA expression was observed in normal brain. Significant agreement between ISH and IHC assays was observed (simple kappa [kappa] estimate=0.358, weighted kappa=0.89, p=0.001). IL-13Ralpha2 protein and mRNA are expressed to significantly higher levels in BSG than in normal brain tissue. Both IHC and ISH represent robust methods to detect expression of the IL-13Ralpha2 receptor in BSG that could represent an important new drug target for treatment of this disease.