Pasteurella multocida toxin (PMT) is a potent mitogen, which is known to activate phospholipase C (PLC) beta by stimulating the alpha-subunit of the heterotrimeric G-protein G(q). PMT also activates RhoA and RhoA-dependent pathways. Using YM-254890, a specific inhibitor of G(q/11), we studied whether activation of RhoA involves other G proteins than G(q/11). YM-254890 inhibited PMT- or muscarinic M3-receptor-mediated stimul-ation of PLCbeta at similar concentrations in HEK 293m3 cells. In these cells, PMT-induced RhoA activation and enhancement of RhoA-dependent luciferase activity were partially inhibited by YM-254890. In Galpha(q/11)-deficient fibroblasts, PMT-induced activation of RhoA, increase in RhoA-dependent luciferase activity and increase in ERK phosphorylation. None of these effects were affected by YM-254890. However, RhoA activation by PMT was inhibited by RGS2, RGS16, lscRGS and dominant negative G(13)(GA), indicating involvement of Galpha(12/13) in the PMT effect on RhoA. In Galpha(12/13) gene deficient cells PMT-induced stimulation of RhoA, luciferase activity and ERK phosphorylation were blocked by YM-254890, indicating the involvement of G(q). Infection with a virus, harboring the gene of Galpha(13) reconstituted increase in RhoA-dependent luciferase activity by PMT even in the presence of YM-254890. The data show that YM-254890 is able to block PMT activation of Galpha(q) and indicate that in addition to Galpha(q) the G proteins Galpha(12/13) are targets of PMT.