Phospholipase C-beta(2) (PLC beta(2)) is activated both by heterotrimeric G protein alpha- and beta gamma- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC beta isozymes with the rank order of PLC beta(2) > PLC beta(3) > or = PLC beta(1). The sensitivity of PLC beta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLC beta(3) > PLC beta(2) > PLC beta(1) for beta(1)gamma(1/2) and PLC beta(2) > PLC beta(1) >>> PLC beta(3) for beta(5)gamma(2). Rac1 and Rac2 were found to be more potent and efficacious activators of PLC beta(2) than was Cdc42Hs. The stimulation of PLC beta(2) by Rho GTPases and G protein beta gamma dimers was additive, suggesting that PLC beta(2) activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC beta(1)-PLC beta(2) enzymes, beta gamma dimers, and Rho GTPases are shown to require different regions of PLC beta(2) to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC beta(2) were sufficient for efficient stimulation by beta gamma, the presence of the putative pleckstrin homology domain of PLC beta(2) was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC beta regulators, which mediate PLC beta isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC beta(2) by extracellular mediators in intact cells.