The β1-adrenergic receptor (β1-AR), the β3-AR, the endothelin A receptor (ETAR) and the ETBR are present on nuclear membranes in adult ventricular myocytes. These nuclear-localized receptors are functional with respect to ligand binding and effector activation. Furthermore, β-AR agonists isoproterenol (β-AR) and BRL37344 (β3-AR), but not Xamoterol (β1-AR), stimulated de novo RNA synthesis, which was PTX sensitive. In contrast, ET-1 decreased the amount of RNA synthesis. Employing enriched nuclear preparations, we investigated which signaling pathway(s) might be involved in GPCR-mediated changes in RNA synthesis. Accordingly, inhibitors of p38 (SB203580), Erk (PD98059, U0126), JNK (SP600125), PI3K (LY294002, Wortmannin) and Akt (Triciribine) were used. Inhibition of Akt, in the presence of isoproterenol, converted the activation of the β-AR from stimulatory to inhibitory in regards to RNA synthesis. Inhibition of Erk, p38 or JNK had no appreciable effect. In contrast, the inhibitory effect of ET-1 was abrogated by Erk inhibition. Western blotting of the enriched nuclear preparations revealed the presence of both the total and phosphorylated forms of Erk and Akt. Furthermore, transcription assays and western blots also confirmed that this signaling cascade is under strong phosphatase control. Treatment of isolated nuclei with microcystin LR, a potent inhibitor of type I and IIA protein serine/threonine phosphatases, almost doubled the observed level of RNA synthesis under basal conditions and following β-AR agonist treatment. Additionally, higher levels of Erk phosphorylation were detected in the presence of microcystin. These results demonstrate that two nuclear-localized GPCRs have markedly different effects upon transcription, and suggest that these two receptor families have distinct roles in the regulation of gene expression in cardiomyocytes.