The hypertrophic response is characterized by increased myofibril/sarcomere organization, induction of the cardiac specific atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2v) genes, and an increase in total cell volume. Theα₁-adrenergic agonist phenylephrine induces both the morphological and biochemical markers of hypertrophy in cultured neonatal rat ventricular cardiomyocytes. Previous studies have suggested a functional requirement for the heterotrimeric G-protein, Gαq, for a subset of the hypertrophic phenotypes. The small GTPases Ras and Rho have also been implicated in phenylephrine-induced hypertrophy. To further delineate the role of Gαq in hypertrophy, a constitutively active mutant of Gαq was transiently transfected in primary rat ventricular cardiomyocytes. This molecule was sufficient to induce ANF-, AP1-, and MLC-2-driven gene expression. Co-transfection of Gαq and dominant negative Ras or dominant negative Raf resulted in dose-dependent inhibition of ANF-driven expression. Both dominant negative Rho, and the Rho inhibitor C3-transferase, also attenuated Gαq- and Ras-induced ANF-driven gene expression. Cells transfected with active Gαq did not show a detectable increase in activation of the mitogen activated protein kinases ERK or SAPK. However, activity of the MAP-kinases appears to be important for Gαq-induced gene expression since the MAP-kinase phosphatase Clone 100 and catalytically inactive SAPK strongly inhibited Gαq-induced ANF expression. Thus, our studies indicate Gαq-induced hypertrophic gene expression requires the small G-proteins Ras and Rho. The data also indicates that Gαq mediated gene expression is dependent on functional MAP-kinases and that multiple signaling pathways contribute to Gαq-mediated cardiac cell hypertrophy.