In myocardial ischemia, pH_i and [ATP] fall, whereas the free [Ca²⁺] and [Mg²⁺] rise. The effects of these changes on cardiac Ca²⁺ release channel (ryanodine receptor) activity were investigated in [³H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg²⁺ and ATP, cardiac Ca²⁺ release channels were half-maximally activated at pH 7.4 by ≈4 μmol/L cytosolic Ca²⁺ and half-maximally inhibited by ≈9 mmol/L cytosolic Ca²⁺. Regulation of channel activity by Ca²⁺ was modulated by Mg²⁺ and ATP. Single-channel activities were more sensitive to a change of cytosolic pH than SR lumenal pH. Reduction in lumenal and/or cytosolic pH from 7.3 to 6.5 and 6.0 resulted in decreased single-channel activities without a change in single-channel conductance. [³H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca²⁺ release channel activity. Mg²⁺ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca²⁺ dependence of binding. In the presence of 5 mmol/L Mg²⁺ and 5 mmol/L β,γ-methyleneadenosine 5′-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca²⁺] for half-maximal [³H]ryanodine binding was increased from 1.9 μmol/L at pH 7.3 to 36 μmol/L at pH 6.5 and to 89 μmol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca²⁺ release channel activity in ischemic myocardium include an altered Ca²⁺ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg²⁺.