In resting muscle, cytoplasmic Mg 2+ is a potent inhibitor of Ca 2+ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites ( I -sites) and to high affinity Ca 2+ sites ( A -sites) thus preventing Ca 2+ activation. We investigate the effects of luminal and cytoplasmic Ca 2+ on Mg 2+ inhibition at the A -sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg 2+ inhibits RyRs at the A -site in the absence of Ca 2+ , indicating that Mg 2+ is an antagonist and does not simply prevent Ca 2+ activation. Cytoplasmic Ca 2+ and Cs + decreased Mg 2+ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca 2+ regulation of Ca 2+ release whereby increasing luminal [Ca 2+ ] decreases the A- site affinity for cytoplasmic Mg 2+ by a noncompetitive, allosteric mechanism that is independent of Ca 2+ flow. Ryanodine increases the Ca 2+ sensitivity of the A- sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca 2+ , indicating that ryanodine activates independently of Ca 2+ . We describe a model for ion binding at the A -sites that predicts that modulation of Mg 2+ inhibition by luminal Ca 2+ is a significant regulator of Ca 2+ release from the SR. We detected coupled gating of RyRs due to luminal Ca 2+ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca 2+ and cytoplasmic Mg 2+ did not compete at the A- sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca 2+ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively “immune” to Ca 2+ emanating from their own pore but sensitive to Ca 2+ from neighboring channels.