RhoA is a small G protein that is implicated in the regulation of the actin cytoskeleton, gene expression, and cell cycle progression, It is activated by many agonists whose receptors are linked to heterotrimeric G proteins, but the mechanisms are incompletely understood. In this study, we show that the constitutively active alpha -subunit of the heterotrimeric G protein G(13) associated with the Rho family guanine nucleotide exchange factor Dbl in NIH 3T3 cells and that this resulted in activation of RhoA. This activation was not seen with wild-type G alpha (13) if Dbl and active G alpha (13) were expressed separately and mixed. In contrast, coexpression of constitutively active G alpha (q) With Dbl did not lead to their association and caused a weak activation of RhoA that was no greater than that observed with wild-type G alpha (q). These findings illustrate that activated G alpha (13) and Dbl can associate in vivo and that this leads to Rho activation.