Tyrosine phosphorylation has been implicated as a means by which neurite outgrowth is regulated. Because paxillin is a tyrosine-phosphorylated protein that may play a role in regulating cell morphology, we examined its expression in neuronal cells and how its tyrosine phosphorylation is related to neurite outgrowth. Paxillin was identified in several neuronal cell lines with an increased level upon differentiation. In SH-SY5Y cells, paxillin was localized along with actin filaments where processes extended from the cell body and in neuritic growth cones. Furthermore, paxillin was tyrosine-phosphorylated in SH-SY5Y cells upon adhesion to laminin. Paxillin tyrosine phosphorylation paralleled that of focal adhesion kinase and occurred as cell spreading, and neurite formation was initiated. Colchicine blocked neurite outgrowth but had no effect on cell spreading or on paxillin or focal adhesion kinase tyrosine phosphorylation. In contrast, cytochalasin D eliminated neurite outgrowth, cell spreading, and the tyrosine phosphorylation of paxillin and focal adhesion kinase. These results show that paxillin is tyrosine-phosphorylated upon integrin ligand binding in neuronal cells. Our findings suggest that paxillin tyrosine phosphorylation is linked to a remodeling of the actin cytoskeleton that leads to cell spreading and neurite formation and thus a differentiated neuronal phenotype.