As a result of increased haemodynamic load, genetic factors or disease, the heart undergoes a hypertrophic response. Genetic manipulation of components of Ca²⁺ regulated gene transcription pathways has revealed that this ion has a fundamental role in stimulating cardiac hypertrophy. However, it is difficult to reconcile how Ca²⁺-sensitive gene transcription can be controlled with great fidelity and specificity independent of the increases in Ca²⁺ that occur during every heart beat. Previously, we have shown that endothelin (ET-1) stimulates InsP₃-dependent Ca²⁺ release in cardiac myocytes (1). ET-1 is also a potent inducer of cardiac hypertrophy. Here we investigated whether ET-1/InsP₃-induced Ca²⁺ release contributes to its pro-hypertrophic action. Experiments were performed using spontaneously contracting primary cultures of neonatal ventricular rat myocytes (NRVM). NRVMs were prepared by collagenase digestion of cardiac ventricles isolated from 1-2 day old Wistar rat pups. Quantitation of atrial natruiretic factor (ANF) by immunofluorescence and real-time PCR, together with cell surface area were used as measures of hypertrophy. For brevity, only the percentage of ANF expressing cells will be presented. All statistics presented are a mean ± SEM of the data generated from myocytes isolated on three or more separate days. Statistical significance was determined by ANOVA and accepted if p < 0.05. To investigate the mechanism of ET-1 induced hypertrophy, 2-APB (10 µM) was first used to inhibit InsP₃-induced Ca²⁺ release. 2-APB decreased the percentage of myocytes expressing ANF following ET-1 stimulation (100 nM, 24 hr) from 54.69±2.5% to 29.85±0.84% (p<0.05). 17.55±4.33% of control, non-stimulated cells expressed ANF. To further demonstrate the InsP₃-dependence of this process, InsP₃-induced Ca²⁺ release was inhibited by adenoviral mediated expression of InsP3- 5’phoshatase. In myocytes expressing 5’phosphatase, ET-1-induced ANF expression (32.33±3.26%) was significantly inhibited when compared to ET-1-stimulated GFP expressing cells (58.32± 4.84%, p<0.05). To determine whether Ca²⁺ entry arising through voltage gated channels was required for ET-1-induced hypertrophy, ET-1 was applied to cells in which voltage channels were blocked with nifedipine (10 µM) and mibefridil (1.8 µM). The percentage of cells expressing ANF was significantly lower in these inhibited cells (11.08±1.66) in comparison to controls (46.41±2.49, p<0.05). Under these conditions, ET-1 significantly increased the percentage of ANF expressing cells (43.46±12, p<0.05). Together, these data demonstrate that InsP₃-induced Ca²⁺ release is required for the induction of hypertrophy as a result of Gq stimulation or increased workload. Furthermore, this provides a mechanism by which gene transcription can be isolated from the increases in cytosolic Ca²⁺ that occur during every heart-beat.