Neuroblastoma is a common pediatric solid tumor that is frequently incurable. Biological risk stratification implicates amplification of the MYCN oncogene as a robust marker for poor prognosis. We have undertaken an analysis of gene expression in MYCN-amplified and non-amplified neuroblastoma tumors based on complementary DNA microarrays to advance our knowledge of the pathogenesis of this aggressive disease. We isolated messenger RNA from tumor cell primary cultures of two INSS stage 4 neuroblastoma patients, one with MYCN amplification and the other with normal MYCN copy number. Relative gene expression was determined after hybridization of the fluorophore-labeled cDNAs to a human cDNA microarray representing over 6,800 unique expressed genes. Using a differential gene expression ratio of 3.5 as a screening criterion, we identified 370 unique genes for further analysis. Of these, 78 were upregulated in the MYCN-amplified neuroblastoma and 292 had decreased expression. Amplification correlated with increased transcript levels of genes associated with cell proliferation, including cell cycle regulatory genes such as CDC2 and CDC28 protein kinase, and the Ki-67 antigen, as well as many other annotated molecules not previously linked to MYCN amplification and several uncharacterized expressed sequence tag sequences. A coherent group of genes was downregulated in the MYCN-amplified cells, including the genes for the class I and class II major histocompatibility complex antigens, the gene for peptide antigen transporter TAP1, and several interferon responsive transcripts. MYCN amplification correlates with a distinct gene expression pattern in neuroblastoma that is consistent with the clinical phenotype of these tumors. Further characterization of this motif should enable the development of new therapies for these difficult tumors.