Molecular mechanisms through which MYCN expression contributes to the malignant phenotype of neuroblastoma are unknown. We performed a genome-widegene expression analysis of 40 well-characterized neuroblastic tumors and 12 cell lines to identify genes and biological pathways associated with MYCN expression. Gene expression was validated by reverse transcription-PCR and immunohistochemistry using tissue arrays. Two hundred twenty-two of 62,839 oligonucleotide probe sets detected expression of genes that were strongly associated with MYCN expression. Differentially expressed genes included examples of known oncogenes, genes associated with neural differentiation, and genes related to cell proliferation. Expression of a subset of these genes was altered after transfection of a neuroblastoma cell line, SK-N-ER, with a MYCN expressing gene construct when protein synthesis was inhibited and have consensus MYCN binding E-box sequences in their promotor regions, suggesting they represent direct targets. Several novel genes/expressed sequences were identified as overexpressed and most likely coamplified with MYCN in a subset of cases. A classification model to identify neuroblastomas with high levels of MYCN expression was developed based on expression profiles. The identification of coexpressed and coamplified genes associated with MYCN overexpression in neuroblastoma suggests biochemical pathways that contribute to the malignant behavior of these tumors and forms a basis for molecular classification.