The mechanisms of action of bacterial toxins have provided novel approaches to elucidating cellular and molecular regulatory mechanisms. In this work the effects of Pasteurella multocida toxin (PMT), cytotoxic necrotizing factor (CNF) from Escherichia coli and the dermonecrotic toxin (DNT) from Bordetella bronchiseptica on tyrosine phosphorylation and cytoskeleton reorganization were investigated in Swiss 3T3 fibroblasts. Recently, tyrosine phosphorylation of the focal adhesion kinase (p125FAK) and the cytoskeleton-associated protein paxillin were identified as early events in the action of diverse signalling molecules that mediate cell growth and differentiation. This work shows for the first time that PMT, CNF and DNT enter cells to stimulate tyrosine phosphorylation of multiple proteins including p125FAK paxillin. The increase in tyrosine phosphorylation of p125FAK and paxillin are accompanied by profound alterations in the organization of the actin cytoskeleton. This study demonstrates that PMT, CNF and DNT induce striking increase in stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. The rho gene products (p21rho), have been implicated in mitogen-stimulated assembly of focal contacts, actin stress fiber formation and tyrosine phosphorylation of p125FAK and paxillin. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21rho function, prevents tyrosine phosphorylation of focal adhesion proteins in response to either PMT, CNF1 or DNT. In addition, here it is shown that CNF1 and DNT do not stimulate PKC activation, inositol phosphate formation or Ca2+ mobilization. Moreover, CNF1 and DNT stimulate DNA synthesis without activation of p42MAPK or p44MAPK providing evidence for a novel p21rho-dependent signalling pathway that leads to entry into S phase of the cell cycle. In conclusion, these findings raise the possibility that tyrosine phosphorylation of p125FAK and paxillin, actin stress fiber formation, focal adhesion assembly and p21rho function may lie in a novel signal transduction pathway targeted by PMT, CNF and DNT.