G13 belongs to the G12 family of heterotrimeric G proteins, whose effectors are poorly defined. The present study was designed to test if phospholipase D (PLD) is regulated by G13 and if Rho-type small GTPases are involved. Expression of the constitutively active Q226L mutant of the alpha-subunit of G13 in COS-7 cells stimulated the activity of a rat brain phospholipase D isozyme (rPLD1) co-expressed in the cells. Wild type Galpha13 was ineffective unless the cells were incubated with AlF4-. rPLD1 was previously shown to be activated by constitutively active V14RhoA in COS-7 cells (Park, S. K., Provost, J. J., Bae, C. D., Ho, W. T., and Exton, J. H. (1997) J. Biol. Chem. 272, 29263-29272). When the endogenous Rho proteins of the cells were inactivated by treatment with C3 exoenzyme from Clostridium botulinum, the ability of Galpha13Q226L to activate rPLD1 was greatly attenuated. Co-transfection of dominant negative N19RhoA and N17Rac-1, but not N17Cdc42Hs or N17Ras, also inhibited the activation. Expression of constitutively active Galphaq in COS-7 cells also activated rPLD1, but constitutively active forms of Galphai2 and Galphas were without effect. These findings support an effector role for PLD in G13 signaling and demonstrate a requirement for Rho GTPases in this response.