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RGS2 and RGS4 were studied for their effects as GTPase activating proteins (GAPs) on receptor-activated Gi in a novel steady-state assay using membranes from Sf9 cells quadruply infected with baculoviruses encoding the m2 muscarinic receptor, Gαi2, Gβ1, and Gγ2. In the presence of the muscarinic agonist carbachol, regulator of G protein signaling 2 (RGS2) and RGS4 each produced up to a 10-fold increase in agonist-dependent GTPase activity. The observed K m for GTP in this system was increased in the presence of RGS4. NaCl and KCl inhibited the GAP activities of both RGS2 and RGS4, although they had no effect on GTPase activity in the absence of RGS proteins. MgCl2 had a complex effect on GTPase activity, with optimal RGS2 and RGS4 GAP activities occurring, respectively, at high micromolar and low millimolar concentrations of free Mg2+. The concentration dependence of RGS GAP activity was assessed, and RGS4 was found to be more potent than RGS2 by up to an order of magnitude. This direct observation confirms a similar difference in potency found when these two RGS proteins were compared for their ability to inhibit signaling downstream of Gi([Heximer et al., 1999][1]). RGS2 yielded Hill coefficients greater than 2.0, suggesting that it may bind in a positively cooperative manner to oligomeric structures containing more than one G protein. Furthermore, RGS4 yielded a bell-shaped dose-dependence under low magnesium (0.5 mM) conditions, which is also consistent with the idea of RGS cooperativity. [1]: #ref-16