We analyzed the effects of Rho GTPases on XSnail2 expression during neural crest (NC) ontogeny in Xenopus laevis embryos. The ectopic expression of both dominant-negative (N−) and constitutively active (V−) Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of XSnail2, XFoxD3, and XSnail1. V14RhoA and N17Rac1 were inhibitory, whereas N19RhoA and V12Rac1 increased NC marker gene expression. In reporter assays using a XSnail2 promoter–green fluorescent protein (GFP) construct (α700BA-GFP), the ectopic expression of V14RhoA, N17Rac1, or the Rac1 inhibitor NSC 23766 decreased reporter expression in NC-neural plate, whereas N19RhoA or the RhoA inhibitor Y27632 and V12Rac1 enhanced it. Similarly, transgenic embryos expressing Rho GTPase mutants and GFP under control of the α700BA promoter displayed variations similar to those observed for ectopic RNA and DNA expression. These results show that Rho GTPases can regulate the expression of XSnail2 during NC ontogeny. Developmental Dynamics 236:2555–2566, 2007. © 2007 Wiley-Liss, Inc.