Rho proteins are members of the Ras superfamily of small GTPases. In higher eukaryotes these proteins play pivotal role ill cell movement, phagocytosis. intracellular transport, cell-adhesion. and maintenance of cell morphology, mainly through the regulation of actin microfilaments. The GTPase TcRhol is the only member of the Rho family described in human protozoan parasite Trypanosoma cruzi. We previously demonstrated that TcRhol is actually required for differentiation of epimastigote to trypomastigote forms during the parasite cell cycle. In the present work, we describe cellular phenotypes induced by TcRhol heterologous expression in NIH 3T3 fibroblasts. The NIH-3T3 lineages expressing the TcRhol-G15V and TcRhol-Q76L mutants displayed decreased levels of migration compared to the control lineage NIH-3T3 pcDNA3.1, a phenotype probably due to distinct cell-substrate adhesion properties expressed by the mutant cell lines. Accordingly, cell-substrate adhesion assays revealed that the mutant cell lines of NIH-3T3 expressing TcRhol-positive dominants constructions present enhanced substrate-adhesion phenotype. Furthermore, similar experiments with T. cruzi expressing TeRho1 mutants also revealed an enhancement of cell attachment. These results suggest that TcRho1 plays a conserved regulatory role in cell-substrate adhesion in both NIH-3T3 fibroblasts and T. cruzi epimastigotes. Taken together, our data corroborate the notion that TcRhol may regulate the substrate-adhesion in T. cruzi, a critical step for successful progression of the parasite life cycle. (c) 2006 Elsevier Inc. All rights reserved.