In cardiac muscle, intracellular Ca(2+) and Mg(2+) are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca(2+)] in the SR ([Ca(2+)](L)) stimulates the Ca(2+) release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg(2+), which has not been regarded as an important regulator of Ca(2+) release. The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca(2+) produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg(2+) was observed, pointing to an important physiological role for luminal Mg(2+) in cardiac muscle. At diastolic [Ca(2+)](C), luminal Mg(2+) inhibition was voltage independent, with K(i) = 45 microM at luminal [Ca(2+)] ([Ca(2+)](L)) = 100 microM. Luminal and cytoplasmic Mg(2+) inhibition was alleviated by increasing [Ca(2+)](L) or [Ca(2+)](C). Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca(2+)-sensing mechanisms on each subunit: activating luminal L-site (40-microM affinity for Mg(2+) and Ca(2+)), cytoplasmic A-site (1.2 microM for Ca(2+) and 60 microM for Mg(2+)), inactivating cytoplasmic I(1)-site (approximately 10 mM for Ca(2+) and Mg(2+)), and I(2)-site (1.2 microM for Ca(2+)). Activation of three or more subunits will cause channel opening. Mg(2+) inhibition occurs primarily by Mg(2+) displacing Ca(2+) from the L- and A-sites, and Mg(2+) fails to open the channel. The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.