We investigated the effects of cytosolic Mg2+ on ryanodine receptor Ca2+ release channel (RyR) of bovine cardiac sarcoplasmic reticulum incorporated into planar lipid bilayers recording single channel activities. Channels were activated by > or = 0.1 microM Ca2+ in the cis solution. At constant Ca2+, application of Mg2+ (0.1-1 mM) to cis side decreased channel activity in a concentration-dependent manner. A half maximal blocking concentration (Kd) was 35 microM and a complete block was obtained at 1 mM. In the presence of 1 mM free Mg2+ in cis solution, the relation between the channel open probability (Po) and concentration of free Ca2+ in cis solution ([Ca2+]cis) shifted to the right, indicating the competition of Mg2+ and Ca2+. Blocking effects of Mg2+ on RyR were antagonized by increasing [Ca2+]cis > or = 0.1 mM. In the presence of 1 m Mg2+ and 1 mM Ca2+ in cis solution, the channel conductance was markedly depressed to approximately 400 pS (n = 7) from 603 +/- 40 pS (mean +/- S.D., n = 22) in the absence of Mg2+, indicating the flickering block. These results show that Mg2+ causes a direct inhibition of RyR in cardiac SR and this inhibition may be mediated through two different mechanisms. A competition of Mg2+ and Ca2+ at a Ca2+ sensitive site on the RyR and a flickery block of the open channel by Mg2+.