Ca(2+)-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mM, skeletal RyRs with Km = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca(2+)-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca(2+)-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca(2+)-activation. Ca(2+)-inhibition in skeletal RyRs was retained in the presence of 500 mM Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mM Cs+ in the solutions fail to demonstrate Ca(2+)-inhibition, while Ca(2+)-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca(2+)-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca(2+)-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.