Calcium (Ca²⁺) is a highly versatile signal that can regulate many different cellular functions. At any moment in time, the intracellular concentration of Ca²⁺ is tightly regulated through a series of ‘on’ and ‘off’ reactions, which either release Ca²⁺ from the sarcoplasmic reticulum (SR) and/or endoplasmic reticulum (ER) into the cytoplasm or remove this signal by a combined action of pumps and exchangers. This cyclical movement of Ca²⁺ underlies the regular contraction of heart myocytes. Modulation of Ca²⁺ circulation (e.g. by the vasoactive peptide endothelin-1 or the β-adrenergic agonist isoproterenol) alters cardiac muscle contractility resulting in inotropic, lusitropic, or chronotropic effects. However, the effects of such stimuli can also initiate disadvantageous responses. For instance, Ca²⁺ dysregulation during disease in humans and other animals can lead to abnormal contraction of the cells and cardiac arrhythmias. We have recently shown that the intracellular distribution of certain of the proteins and organelles involved in Ca²⁺ signalling determines the nature of agonist-induced Ca²⁺ signals in adult rat cardiac myocytes (Mackenzie et al., 2004). We have now extended these studies to determine the relative subcellular distribution of key proteins involved in endothelin-1-stimulated InsP₃-dependent Ca²⁺ release and Ca²⁺-induced Ca²⁺ release in cardiac myocytes. To this end, we have used confocal microscopy to visualise the intracellular distribution of the endothelin A and B receptors (ET_AR and ET_BR), the inositol 1,4,5-triphosphate receptor (InsP₃R), ryanodine receptor (RyR), L-type channel (LTC) and Na/Ca Exchanger (NCX). These studies showed that InsP₃Rs are distributed primarily around the nuclei and under the plasma membrane. This distribution is unlike that observed for the RyR, which was found throughout the cell. LTCs and NCX were distributed on the plasma membrane. ET_ARs were located on the plasma membrane throughout the cell, whereas ET_BRs were found only on the plasma membrane at the polar ends of the cell. In addition, to gain insight into how InsP₃Rs and RyRs are differentially distributed on intracellular Ca⁺² signalling organelles, we have performed confocal immunofluorescence studies with antibodies raised against proteins found exclusively on the ER. These studies indicated that the ER and the SR in cardiac myocytes are distinct organelles, adding a further level of complexity to the understanding of Ca⁺² signalling in these cells.