Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate G(q)and G(i/o)-induced ERK and Akt phosphorylation. To isolate G(q)- and G(i/o)-mediated effects, we exclusively expressed muscarinic M(2) or M(3) receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in M(2)-/M(3)- expressing cells through carbachol stimulation. In co-expressions, M(3)/G(q)-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. M(2)/G(i/o) induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on M(2)/G(i/o)-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of G(q)- and G(i/o)-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards G(q) or G(i/o) proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.