Gβγ dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gβγ in those signaling processes mediated by small GTP-binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gβγ to immobilized GST-Rho fusion proteins. In addition, brain Gβγ, but not transducin Gβγ, was able to inhibit GTPγS binding to GST-Rho in a concentration-dependent manner. GTPγS binding to GST-Rac was also decreased by brain Gβγ whereas nucleotide binding to GST-Cdc42 was not changed. We conclude that Gβγ dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.