Signalling pathways using heterotrimeric guanine-nucleotide-binding- proteins (G proteins) trigger physiological responses elicited by hormones, neurotransmitters and sensory stimuli. GTP binding activates G proteins by dissociating Gα from Gβγ subunits, and GTP hydrolysis by Gα 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 Gα subunits in vitro, suggesting that GTPase-activating proteins (GAPs) deactivate Gα subunits. Here we report that RGS (for regulator of G-protein signalling) proteins are GAPs for Gα subunits. RGS1, RGS4 and GAIP (for Gα-interacting protein) bind specifically and tightly to Gα(i) and Gα(o) in cell membranes treated with GDP and AlF4/-, and are GAPs for Gα(i), Gα(o), and transducin α- subunits, but not for Gα(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.