The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 mu M Ca2+, Ca2+ release from Ca-45(2+)-loaded SR vesicles prepared from porcine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca2+-activated [H-3]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR Ca-45(2+) release or [H-3]ryanpdine binding. Ca2+-activated [H-3]ryanodine binding was increased similar to 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [H-3]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate- containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (less than or equal to 10 mM). Transitions to this putative Ca2+-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.