Cardiac myocytes contract in situ in response to electrical stimuli from the sinoatrial (SA) node. Contraction is facilitated by Ca²⁺-induced Ca²⁺ release (CICR) through ryanodine receptors (RyRs) on the surface of the sarcoplasmic reticulum (SR). Cardiac myocytes also express inositol 1, 4, 5-trisphosphate receptors (InsP₃Rs), albeit at a 100-fold lower abundance than RyRs. In response to stimulation of PLC-coupled receptors such as the endothelin-1 receptor (ET-1R), or InsP₃ itself, we have previously observed Ca²⁺ release in adult rat ventricular and atrial myocytes. Unlike the global Ca²⁺ changes observed during excitation-contraction coupling through RyRs, Ca²⁺ liberated via InsP₃Rs is more restricted in its spatial distribution. As well as inducing an inotropic effect, however, InsP₃-induced Ca²⁺ release can promote arrhythmogenic Ca²⁺ signals in cardiac myocytes. A role for InsP₃-induced Ca²⁺ release in hypertrophic signalling has also recently been suggested. Since rat ventricular neonatal myocytes are a widely used and accepted model for analysis of cardiac myocyte signalling pathways, we have now investigated the mechanisms involved in generating Ca²⁺ signals in these cells. To this end, neonatal ventricular myocytes were isolated from 1-2 day old Wistar rats and cultured to form a spontaneously contracting monolayer. Confocal Ca²⁺ imaging of these myocytes demonstrated small Ca²⁺ release events that were primarily perinuclear, which occurred independent of whole cell Ca²⁺ signals controlling contraction. These Ca²⁺ release events were sensitive to inhibition by the RyR antagonist ryanodine but not the InsP₃R antagonist 2-aminoethoxy diphenylborate (2-APB). In spontaneously contracting cells, application of ET-1 induced an increase in frequency but not amplitude of the myocyte Ca²⁺ signals. Application of ET-1 in cells in which CICR was abolished stimulated calcium release, which was concentrated in the perinuclear region. This perinuclear Ca²⁺ transient was also observed in cells exposed to a membrane permeable IP₃ ester. This ET-1-induced Ca²⁺ signal was abolished following pre-treatment with 2-APB. Together, these data indicate that neonatal myocytes express both functional InsP₃Rs and RyRs, which can act independently to generate spatially distinct Ca²⁺ release events.