POLYPEPTIDES that define a protein family termed RGS (for regulators of G-protein signalling) are encoded by the SST2 gene of the yeast Saccharomyces cerevisiae, the EGL-10 gene of the nematode Caenorhabdatis elegans, and several related mammalian genes. Genetic studies in invertebrates and mammalian cell-transfection experiments indicate that RGS proteins negatively regulate signalling pathways involving seven transmembrane receptors and heterotrimeric G proteins(1-3). However, the biochemical mechanism by which RGS proteins control these pathways is unknown. Here we report the characterization of human RGS10, a member of this protein family. Co-immunoprecipitation studies demonstrate that RGS10 associates specifically with the activated forms of two related G-protein subunits, G alpha(i3), and G alpha(z) but fails to interact with the structurally and functionally distinct G alpha(s) subunit. In vitro assays with purified proteins indicate that RGS10 increases potently and selectively the GTP hydrolytic activity of several members of the G alpha(i) family, including G alpha(i3), G alpha z and G alpha(o). These results demonstrate that RGS proteins can attenuate signalling pathways involving heterotrimeric G proteins by serving as GTPase-activating proteins for specific types of G alpha subunits.