In heart failure, cardiac ryanodine receptors (RyR2) become abnormally active or “leaky” and are unable to remain closed during diastole (1). This results in unwanted irregular contractility. Inhibition of channel gating by Mg2+ helps prevent inappropriate activation of RyR2. Aberrant Zn2+-homeostasis is associated with chronic heart failure (2) raising the question to the role of Zn2+ in regulating RyR2 function. Here we have studied how Zn2+ influences the gating of single native RyR2 channels. Sheep RyR2 channels were incorporated into planar phosphatidylethanolamine lipid bilayers under voltage-clamp conditions as previously described (3). Unless stated, single-channel recordings were obtained with 250mM HEPES, 80mM Tris, 10µM free Ca2+, pH 7.2, on the cis (cytoplasmic) side and 250mM glutamic acid, 10mM HEPES, pH to 7.2 with Ca(OH)2 (free [Ca2+] approximately 50mM) on the trans (luminal) side of the bilayer. The luminal chamber was voltage-clamped at ground. Open probability (Po) was determined over 3 min of continuous recording. Student’s t-test was used to assess the difference between mean values. In cardiomyocytes the resting intracellular Zn2+ concentration is reported to be at 100pM (4). Interestingly the addition of 100pM Zn2+ to the cytosolic face of the channel significantly increased RyR2 activity from 0.10 ± 0.03 to 0.45 ± 0.04 (S.E.M; n=4; P<0.05). Under these conditions, channel activation was still dependent on the presence of activating levels of cytosolic Ca2+. At concentrations of free Zn2+ reported to occur under pathophysiological conditions (>1nM) Zn2+ became the main activating ligand and the dependency on Ca2+ was removed. In line with previous studies (5), we show that exposure of RyR2 to 1mM cytosolic Mg2+ reduced channel Po from 0.34 ± 0.05 to 0.05 ± 0.01 (S.E.M; n=2). In the continued presence of 1mM Mg2+ the subsequent addition of 100pM Zn2+ to the cis chamber caused a marked increase in channel Po (0.36 ± 0.04; S.E.M, n=2). This suggests that Zn2+ may play a key role in regulating channel function enabling RyR2 to operate under conditions of systole. When the cytosolic [Ca2+] was lowered to 100nM, our preliminary data (n=1) reveal that in the continued presence of 1mM Mg2+ the subsequent addition of 1nM cytosolic Zn2+ caused channel Po to increase from 0.06 to 0.19. Under these conditions RyR2 channels are expected to remain closed. This suggests that pathophysiological levels of Zn2+ may lead to irregular opening of RyR2 channels during diastole. We propose that Zn2+ plays a key role in shaping intracellular Ca2+-dynamics through modulation of RyR2 and that pathological perturbations in Zn2+-homeostasis may lead to inappropriate release of Ca2+ leading to the progression of heart failure and fatal arrhythmias.