During the diastolic depolarization phase of the sinoatrial (SA) node action potential, spontaneous localised Ca2+ release events (Ca2+ sparks) from the sarcoplasmic reticulum (SR) occur. Elevation of subcellular Ca2+ as a result of these Ca2+ sparks activates the forward mode of the Na+-Ca2+ exchanger (NCX1), which generates an inward current and accelerates late diastolic depolarization and helps bring the membrane potential to the threshold potential for the firing of an action potential (1). This is referred to as the Ca2+ clock mechanism of SA node automaticity. In ventricular myocytes, Ca2+ sparks can occur during diastole, but they serve no known function other than to relieve the SR of Ca2+ overload. Consequently, Ca2+ spark characteristics in the two cell types may differ and the aim of this study was to investigate this.Single myocytes were isolated by enzymatic digestion of the SA node and ventricles of hearts taken from adult male Wistar rats weighing approximately 200-225 g. All procedures were in accordance with the Animals (Scientific Procedures) Act 1986. Changes in [Ca2+]i were detected by loading cells with 5 µM Fluo-8 AM. Cells were bathed in Tyrode’s solution (containing in mM: 140 NaCl, 5.4 KCl, 1 MgCl2, 5 HEPES, 5.5 glucose, 1.8 CaCl2). Confocal Ca2+ imaging was performed using an Andor Revolution XD confocal system with a Yokogawa CSU spinning disk. Ca2+ sparks were analysed from x-y image stacks using recently published xySpark software from Steele and Steele (2).Mean Ca2+ spark amplitude (ΔF/F0) was 0.39 ± 0.03 in ventricular myocytes (n = 8) and 0.67 ± 0.08 in SA node myocytes (p<0.05, n=17), an increase of 72%. Average Ca2+ spark frequency (measured as sparks/1000 µm2 /second) was also greater: 14.7 ± 3.5 in ventricular myocytes and 31.8 ± 5.4 in SA node myocytes (p<0.05). Mean spatial width and Ca2+ spark duration were unchanged between SA node and ventricular myocytes.The results show that a greater Ca2+ release occurs from the SR during Ca2+ sparks in SA node myocytes than in ventricular myocytes. A greater subcellular SR Ca2+ release in SA node myocytes will facilitate activation of NCX1, which helps drive the late diastolic depolarization phase of the SA node action potential.