SIGNALLING pathways using heterotrimeric guanine-nucleotide-binding-proteins (G proteins) trigger physiological responses elicited by hormones, neurotransmitters and sensory stimuli(1,2) GTP binding activates G proteins by dissociating G alpha from G beta gamma subunits, and GTP hydrolysis by G alpha subunits deactivates G proteins by allowing heterotrimers to reform. However, deactivation of G-protein signalling pathways in vivo can occur 10- to 100-fold faster than the rate of GTP hydrolysis of G alpha subunits in vitro(3-8), suggesting that GTPase-activating proteins (GAPs) deactivate G alpha subunits. Here we report that RGS(9,10) (for regulator of G-protein signalling) proteins are GAPs for G alpha subunits. RGS1, RGS4 and GAIP (for G alpha-interacting protein(17)) bind specifically and tightly to G alpha(i) and G alpha(o) in cell membranes treated with GDP and AIF(4)(-), and are GAPs for G alpha(i), G alpha(o) and transducin alpha-subunits, but not for G alpha(s). Thus, these RGS proteins are likely to regulate a subset of the G-protein signalling pathways in mammalian cells. Our results provide insight into the mechanisms that govern the duration and specificity of physiological responses elicited by G-protein-mediated signalling pathways.