Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the alpha(3) integrin subunit of alpha(3)beta(1) integrin is required for alpha(3)beta(1) integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) alpha subunit and the cytoplasmic sequence of the alpha(3) integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic alpha(3) tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic alpha(3) sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-alpha(3) can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase alpha.