The role of inositol 1,4,5-trisphosphate (InsP₃) in cardiac myocyte function is unclear and controversial. Although agonists that activate InsP₃ generation are positive inotropic agents in the heart and have been implicated in various cardiac pathologies, it is unclear whether InsP₃ is responsible for any of their effects. We investigated the expression and function of InsP₃ receptors (InsP₃Rs) in rat ventricular and atrial cardiac myocytes. Rats were killed in accordance with schedule 1 Home Office regulation, and myocytes were enzymatically isolated using a standard procedure (Mackenzie et al., 2001). Cells were electrically paced at 0.3 Hz using field electrodes and calcium changes were monitored using single cell photometry or confocal imaging. Stimulation with a membrane-permeant form of InsP₃ (InsP₃ ester; 2 μM) caused a modest positive inotropic response in both atrial and ventricular myocytes. The most prominent effect of the InsP₃ ester was to provoke the occurrence of spontaneous diastolic calcium transients. These events were rare in control recordings. For both cell types, the spontaneous calcium transients evoked by InsP₃ ester were prevented by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate (2-APB; 2 μM). Endothelin-1 (ET-1) acts via multiple signalling pathways to modulate the inotropic status of the heart and is known to cause arryhthmias. ET-1 stimulation of atrial or ventricular myocytes caused a significant positive inotropic effect and also triggered substantial numbers of spontaneous calcium transients. These events were inhibited by 2-APB, suggesting that InsP₃Rs were responsible for the increased automaticity. The patterns of spontaneous calcium transients were similar in InsP₃ ester- or ET-1-treated cells. For both stimuli, the spontaneous diastolic calcium signals largely occurred on the falling phase of the previous action potential-evoked calcium transient. In addition, the spontaneous calcium signals appeared to be due to the triggering of calcium sparks and/or action potentials. Beta-adrenergic stimulation (using isoproterenol; 100 nM) also caused spontaneous calcium transients, but the characteristics of these signals were distinct from those evoked by InsP₃ ester or ET-1. The spontaneous calcium transients evoked by isoproterenol typically occurred >1s after the previous action potential when the calcium level had recovered to diastolic levels. Furthermore, the events caused by isoproterenol were largely observed as calcium waves with few action potentials. 2-APB did not affect the characteristics of isoproterenol-induced spontaneous calcium transients. Our data support a role for InsP₃ in mediating a modest inotropic effect in the heart. The most prominent effect of InsP₃R stimulation appears to be the triggering of pro-arrhythmic spontaneous calcium transients. Direct stimulation of InsP₃Rs in atrial or ventricular myocytes mimics the pattern of response observed with the natural cardioactive hormone ET-1. The properties and mechanism of the spontaneous calcium signals evoked by InsP₃R- and ET-1-stimulation appear to be distinct from those triggered by increases in cyclic AMP.