We identified a Rho guanine exchange factor (GEF) expressed as two splice variants, which differ only in either having or lacking a Postsynaptic density 95, Disk large, Zona occludens-1 (PDZ) motif. The PDZ adaptor protein synectin bound the longer splice variant, Syx1, which was targeted to the plasma membrane in a synectin-dependent manner. The shorter variant, Syx2, was diffusely distributed in the cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed similar differences between the spatial patterns of active RhoA in Syx1 versus Syx2-expressing cells. Expression of Syx1 augmented endothelial cell (EC) migration and tube formation, whereas Syx2 expression did not. It appears, therefore, that synectin-dependent targeting of Syx is critical to its contribution to these EC functions. Although agonist-stimulated global RhoA activity was similar in Syx1- and Syx2-expressing cells, basal RhoA activity was surprisingly higher in the latter. Out of 23 cell types, we found a significant level of endogenous Syx2 expression only in brain tumor cells, which also exhibited high basal RhoA activity. We found that the activity level of JNK, which mediates transcriptional regulation downstream of RhoA, is elevated in a Syx2-dependent manner in these cells, possibly contributing to their tumorigenicity.