Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein Galpha(12). PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of Galpha(12) with the Aalpha subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that Galpha(12) stimulated phosphatase activity (J Biol Chem 279: 54983-54986, 2004). We now show in substrate-velocity analysis using purified PP2A that V(max) was stimulated 3- to 4-fold by glutathione transferase (GST)-Galpha(12) with little effect on K(m) values. To identify the binding domains mediating the Aalpha-Galpha(12) interaction, an extensive mutational analysis was performed. Well-characterized mutations of Aalpha were expressed in vitro and tested for binding to GST-Galpha(12) in pull-down assays. Galpha(12) binds to Aalpha along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where Aalpha binds to Galpha(12), a series of 61 Galpha(12) mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant Galpha(12) proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-Aalpha in pull-down assays. The Aalpha binding sites were localized to regions near the N and C termini of Galpha(12). The expression of constitutively activated Galpha(12) (QLalpha(12)) in Madin Darby canine kidney cells stimulated PP2A activity as determined by decreased phosphorylation of tyrosine 307 on the catalytic subunit. Based on crystal structures of Galpha(12) and PP2A Aalpha, a model describing the binding surfaces and potential mechanisms of Galpha(12)-mediated PP2A activation is presented.