In previous studies, we have demonstrated that RhoA/B-dependent signaling regulates TGFbeta-induced rapid actin reorganization in Swiss 3T3 fibroblasts. Here we report that TGFbeta regulates long-term actin remodeling by increasing the steady-state mRNA levels of the RhoB gene in mouse Swiss 3T3 fibroblasts and human hepatoma HepG2 cells. We show that this regulation is specific for the RhoB gene and is facilitated by enhanced activity of the RhoB promoter. Adenovirus-mediated gene transfer of Smad2 and Smad3 in Swiss 3T3 fibroblasts induced transcription of the endogenous RhoB gene but not the RhoA gene. Interestingly, in JEG-3 choriocarcinoma cells that lack endogenous Smad3, TGFbeta-induced transcriptional up-regulation of the RhoB gene was not observed, but it was restored by adenoviral Smad3 overexpression. In addition, Smad2 and Smad3 triggered activation of RhoA and RhoB GTPases and long-term actin reorganization in Swiss 3T3 fibroblasts. Finally, Smad3, and to a lesser extent Smad2, induced transcription of the alpha-smooth muscle actin (alpha-SMA) gene, and enhanced the incorporation of alpha-SMA into microfilaments in Swiss 3T3 fibroblasts. These data reveal a novel mechanism of cross-talk between the classical TGFbeta/Smad pathway and Rho GTPases, regulating the rapid and the long-term actin reorganization that may control the fibroblast-myofibroblast differentiation program.