The cardiac muscle sarcoplasmic reticulum Ca²⁺ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca²⁺ concentrations and inactivated by millimolar cytoplasmic Ca²⁺ concentrations. The effects of sarcoplasmic reticulum lumenal Ca²⁺ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca²⁺ concentrations, lumenal-to-cytosolic Ca²⁺ fluxes ≥0.25 pA activated the channel. At the maximally activating cytosolic Ca²⁺ concentration of 4μM, lumenal Ca²⁺ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca²⁺ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca²⁺-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca²⁺-activated channel activities, suggesting that it lowered Ca²⁺ concentrations at cytosolic Ca²⁺-inactivating sites. Regulation of channel activities by lumenal Ca²⁺ could be also observed in the absence of caffeine and in the presence of 5mM MgATP. These results suggest that lumenal Ca²⁺ can regulate cardiac Ca²⁺ release channel activity by passing through the open channel and binding to the channel's cytosolic Ca²⁺ activation and inactivation sites.