N-MYC is a transcription factor that plays an important role in cellular survival in neuroblastoma, and amplification of the N-MYC oncogene is the primary adverse prognostic indicator for neuroblastoma. Focal adhesion kinase (FAK) is a survival factor that has been shown to be overexpressed in many types of human cancers. In this study, we investigated the role of N-MYC regulation of FAK expression in neuroblastoma. We first found a correlation between N-MYC and FAK expression in neuroblastoma. Real time quantitative PCR demonstrated an increase in FAK mRNA abundance in the N-MYC-amplified IMR-32 compared with the nonamplified SK-N-AS neuroblastoma cell lines. FAK protein expression also correlated positively with N-MYC expression in the N-MYC-amplified IMR-32 versus nonamplified SK-N-AS neuroblastoma cell lines. The same results were seen with the isogenic N-MYC- (Tet+) and N-MYC+(Tet -) neuroblastoma cell lines. Promoter-reporter assays showed that activity of the FAK promoter was increased in the N-MYC-amplified IMR-32 cell line, in the N-MYC-transfected SK-N-AS nonamplified cell line, and in the isogenic N-MYC+ (Tet-) neuroblastoma cell lines compared with the nonamplified and N-MYC-nonexpressing cell lines. We also identified two N-MYC binding sites in the FAK promoter sequence and showed binding of N-MYC transcription factor to the FAK promoter through electrophoretic mobility shift, chromatin immunoprecipitation, and dual luciferase assays. Finally down-regulation of FAK expression in N-MYC-inducible neuroblastoma cell lines with FAK small interfering RNA or a dominant-negative FAK inhibitor (AdFAK-CD) significantly decreased viability and increased apoptosis in the N-MYC - (Tet+) cells compared with the isogenic N-MYC - (Tet+) cells, demonstrating the biological significance of FAK overexpression in the N-MYC-expressing cell lines. This is the first report linking N-MYC and FAK in neuroblastoma, and it clearly demonstrates that N-MYC induces FAK expression. The results indicate that N-MYC regulation of FAK expression can control cellular functions in isogenic N-MYC-/+ (Tet+/-) neuroblastoma cell lines. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.