Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for alpha subunits of the Gi and/or Gq class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGS/Galpha protein contact face, reduce binding affinity and inhibition by PIP3. Calmodulin binds all RGS proteins tested in a Ca(2+)-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca2+/calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP3 act as allosteric inhibitors of RGS proteins, and Ca2+/calmodulin competition with PA and PIP3 may provide an intracellular mechanism for feedback regulation of Ca2+ signaling evoked by G-protein-coupled agonists.