The metastatic potential of undifferentiated neuroblastomas is typically lost when differentiation into ganglioneuroblastomas occurs spontaneously or is induced. Cell adhesion may play a role in metastasis, and we have shown recently that expression of integrin αvβ5 protein and mRNA is up-regulated in ganglioneuroblastomas in vivo. To investigate whether interactions of αvβ5 with matrix components play a role in the loss of metastatic potential, we used immunohistochemical and in situ hybridization to analyze neuroblastic tumors at various stages of differentiation for expression of the αβ5 ligands, vitronectin and osteopontin, and determined the ability of vitronectin to promote attachment and neurite outgrowth in vitro in a retinoic-acid-differentiated neuroblastoma cell model. We found that vitronectin, but not osteopontin, was expressed in 5 of 5 ganglioneuroblastomas but was absent or weakly expressed in 6 of 6 undifferentiated neuroblastomas. Neuronal cell vitronectin was detected in 7 of 9 ganglioneuromas, 5 of 8 peripheral ganglia, and 14 of 21 adrenal gland medullae, confirming expression of vitronectin in mature peripheral neurons. In vitro, vitronectin promoted attachment of both undifferentiated and retinoic-acid-differentiated neuroblastoma cells, which was inhibited 20 and 60%, respectively, by monoclonal antibody anti-integrin αvβ5. Vitronectin-promoted neurite outgrowth of retinoic-acid-differentiated neuroblasroma cells was not inhibited by monoclonal antibody anti-αvβ5. These data suggest that the synthesis of vitronectin and the ability of integrin αvβ5 to mediate vitronectin adhesion on retinoic acid-differentiated neuroblastoma cells may promote differentiation of neuroblastoma cells in vivo.