The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain G(α) subunits in vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the G(i) and G(q) subclasses of G(α) subunits but not with G(12α) or G(sα). Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient G(iα) subunit (G(iα2)Q204L) resulted in the translocation of both wild type RGS4 and a non-G(iα)-binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by G-protein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.