The identification of molecular signatures characteristic of tumor cells that are capable of metastatic spread is required for the development of therapeutic interventions to abrogate this lethal process. To facilitate this, we have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 are spontaneously occurring sublines of the MDA-MB-435 cell breast tumor cell line that exhibit phenotypic differences in growth, invasion, and metastatic efficiency in athymic mice. In this study, transcriptional profiles of these cell lines were created using oligonucleotide microarrays representing over 12,000 genes. Intensity modeling and hierarchical clustering analysis identified a 171-gene expression signature that correlated with metastatic phenotype and highlighted several GTPase signaling components. Restoration of one of these GTPases, deleted in liver cancer-1 (DLC-1), in metastatic M4A4 cells to levels observed in the nonmetastatic NM2C5 cell line resulted in the inhibition of migration and invasion in vitro and a significant reduction in the ability of these cells to form pulmonary metastases in athymic mice. These studies show the utility of expression profiling, in an appropriate experimental system, to identify genetic determinants of metastatic sufficiency. The finding that DLC-1 can act as a metastasis-suppressor gene supports an influential role for GTPase signaling in tumor progression.