Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca 2+ and Mg 2+ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca 2+-and Mg 2+-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca 2+ activation (K a = 4 μM) and inhibition by cytoplasmic Mg 2+ (Ki = 10 μM at 100 nM Ca 2+) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca 2+, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg 2+ inhibition than those from sheep and rat. The K a values for luminal Ca 2+ activation were similar in the three species (35 μM for human, 12 μM for sheep, and 10 μM for rat). From the relationship between open probability and luminal [Ca 2+], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg 2+ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca 2+ and Mg 2+ only occurred when cytoplasmic [Ca 2+] was <3 μM. The activation response of RyR2 to luminal and cytoplasmic Ca 2+ was strongly dependent on the Mg 2+ concentration. Addition of physiological levels (1 mM) of Mg 2+ raised the K a for cytoplasmic Ca 2+ to 30 μM (human and sheep) or 90 μM (rat) and raised the K a for luminal Ca 2+ to ~1 mM in all species. This is the first report of the regulation by Ca 2+ and Mg 2+ of native RyR2 receptor activity from healthy human hearts.