The roles of the Ca²⁺-mobilising messenger inositol 1,4,5-trisphosphate (InsP₃) in heart are unclear, although many hormones activate InsP₃ production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca²⁺ release mediated by InsP₃ receptors (InsP₃Rs) [1–3]. In the present study, we examined the expression and subcellular localisation of InsP₃R isoforms, and investigated their potential role in modulating excitation–contraction coupling (EC coupling). Western, PCR and InsP₃-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP₃Rs, with approximately sixfold higher levels of InsP₃Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP₃Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP₃ ester, which enters cells and directly activates InsP₃Rs, caused the appearance of spontaneous Ca²⁺-release events. In addition, in paced cells, the InsP₃ ester evoked an increase in the amplitudes of action potential-evoked Ca²⁺ transients. These data indicate that atrial cardiomyocytes express functional InsP₃Rs, and that these channels could modulate EC coupling.