Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2–RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited G_q/11-mediated phospholipase C β stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited G_q/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both G_q/11 and G_i/o signaling (carbachol-induced cAMP inhibition). G_s signaling was unaffected, and, contrary to reports in other cell lines, RGS2–RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced G_q/11 signaling (α₁-adrenergic stimulation or Gα_q^* overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of G_q/11 signaling (RGS3–RGS5) were unchanged. Endogenous RGS2 (but not RGS3–RGS5) expression was also up-regulated in cells with enhanced AC signaling (β-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of G_q/11 signaling that could potentially become a promising target for ameliorating G_q/11-mediated signaling and growth.