Epithelial cell scattering encompasses the dissolution of intercellular junctions, cell-cell dissociation, cell spreading, and motility. The Rac1 and ARF6 GTPases have been shown to regulate one or more of these aforementioned processes. In fact, activated Rac1 has been shown to promote cell-cell adhesion as well as to enhance cell motility, leading to conflicting reports on the effect of Rac1 activation on epithelial cell motility. In this study, we have examined the activation profiles of endogenous Rac1 and ARF6 during the sequential stages of epithelial cell scattering. Using Madin-Darby canine kidney cells treated with hepatocyte growth factor/scatter factor or cell lines stably expressing activated v-Src, we show that Rac1 and ARF6 exhibit distinct activation profiles during cell scattering. We have found that an initial ARF6-dependent decrease in the levels of Rac1-GTP is necessary to induce cell-cell dissociation. This is followed by a steady increase in Rac1 and ARF6 activation and cell migration. In sum, this study documents the progression of ARF6 and Rac1 activities during epithelial cell scattering.