The small GTP-binding protein Rho rapidly stimulates the formation of focal adhesions and actin stress fibres when microinjected into serumstarved Swiss 3T3 fibroblasts. This response is inhibited by tyrosine kinase inhibitors. Addition of growth factors such as lysophosphatidic acid and bombesin to Swiss 3T3 cells stimulates a similar response, which is dependent on endogenous Rho proteins. To investigate signalling events regulated by Rho, we have scrape loaded Rho into serumstarved cells. Activated Rho stimulates the tyrosine phosphorylation of a number of proteins, including three proteins known to localise to focal adhesions, pp125^FAK, p130 and paxillin. Rhoinduced phosphorylation of pp125^FAK, p130 and paxillin is observed in the absence of stress fibre formation and is, therefore, independent of Rhoinduced actin polymerisation. We propose that the tyrosine kinase, pp125^FAK, and the putative adapter proteins, paxillin and p130, are components of a Rhoregulated signal transduction pathway, and that these protein tyrosine phosphorylation events are likely to be important for the regulation of focal adhesion formation.