As a result of increased haemodynamic load, genetic factors or disease, the heart undergoes a hypertrophic response. Genetic manipulation of components of various Ca2+ regulated gene transcription pathways has revealed that this ion has a fundamental role in stimulating cardiac hypertrophy. Previously, we have shown that Endothelin (ET-1) stimulates InsP3-dependent Ca2+ release in atrial and ventricular myocytes. ET-1 is also a potent inducer of cardiac hypertrophy. Here we investigated whether InsP3- induced Ca2+ release (IICR) contributes to the pro-hypertrophic action of ET-1. Experiments were performed using spontaneously contracting monolayers of primary cultures of neonatal ventricular rat myocytes (NRVM). Quantitation of atrial natruiretic factor (ANF) by immunofluorescence and real-time PCR, together with cell surface area were used as measures of hypertrophy. To investigate the mechanism underlying ET-1 induced hypertrophy, 2-APB or adenoviral mediated expression of InsP3- 5’phoshatase were used to inhibit IICR. Both 2-APB and expression of InsP3- 5’phoshatase significantly decreased the percentage of myocytes expressing ANF following ET-1 stimulation. To determine whether excitation-contraction (EC) coupling was required for ET-1-induced hypertrophy, ET-1 was applied to NRVMs in which voltage gated channels were blocked with nifedipine (L-type Ca2+ channel antagonist) and mibefradil (T-type Ca2+ channel antagonist). Under these conditions, ET-1 significantly increased the percentage of ANF expressing cells in an InsP3-dependent manner, demonstrating that IICR can induce hypertrophy independently of EC-coupling. However, the percentage of cells expressing ANF was significantly lower in these inhibited cells in comparison to controls. To determine whether hypertrophy induced by increased EC-coupling required IICR, BayK 8644 (L-type Ca2+ channel agonist) or isoproterenol (IsoP, β-adrenergic agonist) were used. Both agonists significantly increased the percentage of cells expressing ANF in an InsP3-dependent manner. To investigate the mechanism by which increased EC-coupling/workload induces hypertrophy, antagonists of ET-1 binding, production and secretion were used – BQ123, Phosphoramidon and Primaquine, respectively. All three antagonists significantly decreased the percentage of NRVMs expressing ANF following BayK 8644 or IsoP stimulation, suggesting that increased workload increases ET-1 production and secretion from myocytes. The resultant paracrine/autocrine effect of ET-1 leads to IICR and the induction of hypertrophy. Together, these data demonstrate that IICR is required for the induction of hypertrophy as a result of Gq stimulation or increased workload. Furthermore, this provides a mechanism by which Ca2+ regulated gene transcription can be isolated from the increases in cytosolic Ca2+ that occur during every heart-beat.