Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that α4 and α5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either α4β1- or α5β1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that α5β1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for α5β1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, α4β1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-α overexpression inhibited α4β1-stimulated NB motility and Src activation consistent with α4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In α4 shRNA-expressing NB cells, α4β1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated α4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that α4β1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during α5β1-mediated NB migration and support the evaluation of inhibitors to α4, Src and FAK in the control of NB tumor progression. © 2008 Nature Publishing Group All rights reserved.