Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine nucleotide exchange factor that can activate RhoA and/or Cdc42 in a cell specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA-PRK and RhoA-ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells, and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain (MLC), and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of MLC expression by siRNAs impairs Dbs focus formation, thus establishing a direct link between actino-myosin contraction and RhoGEF transformation.