Low grade astrocytomas are the most common CNS tumors in neurofibromatosis type 1(NF1) patients. While most are classic pilocytic astrocytomas (PA), some are difficult to classify, and have been termed "low grade astrocytoma subtype indeterminate" (LGSI). Some of these tumors exhibit peculiar morphologies, including plump cytoplasmic processes and macronucleoli. In the current study we performed electron microscopy, followed by gene expression, immunohistochemicai and western blot analyses in an effort to identify biological differences underlying phenotypic variation in NF1-associated low grade astrocytoma. Electron microscopy demonstrated intermediate filaments and frequent Rosenthal fiber material in both PA and LGSI. Dense core granules and/or aligned microtubules were present in the LGSI group (2 of 3 cases) and in the PA group (1 of 10 cases). Analysis of global gene expression data obtained using Affymetrix HG-U133 Plus2.0 chips (5 PA, 1 LGSI), and western blot analysis for phospho-S6 (1 LGSI, 2 PA) demonstrated a gene expression profile reflecting "neuronal differentiation" and increased phospho-S6 immunoreactivity consistent with mTOR activation in the LGSI compared with PA. These findings were confirmed by immunohistochemistry for neuronal markers, as well as combined phospho-S6/ phospho-p70S6K immunoreactivity in 4 (of 4) LGSI vs. 5 (of 13) NF1-associated PA (p=0.02), and 13 (of 39) sporadic PA. Phospho-ERK immunoreactivity was uniformly present in PA and LGSI groups, while BRAF duplication was absent by FISH in 8 NF1-associated low grade astrocytomas. In summary, differential expression of neuronal-related genes and increased mTOR activation may underlie phenotypic variations in NF1-associated low grade astrocytomas.