In this study terminal cisternae vesicles from rabbit skeletal muscle were fused into planar bilayers and the effect of calmodulin on single Ca2+ release channel currents was investigated. In the presence of 10(-7) and 10(-9) M free [Ca2+], nanomolar concentrations of calmodulin activated the channel by increasing the open probability of single-channel events in a dose dependent manner. The activatory effect of calmodulin was reversed by 10 microM ruthenium red. At 10(-5) M free [Ca2+], calmodulin (0.1-1 microM) inhibited channel activity. Calmodulin overlays were carried out using concentrations of Ca2+ similar to those used for the planar lipid bilayer assays. In the presence of 10(-7) M [Ca2+], calmodulin bound to the ryanodine receptor, to a region defined by residues 2937-3225 and 3546-3655. These results suggest that calmodulin may activate the Ca(2+)-release channel (ryanodine-receptor) by interacting with binding sites localized in the central portion of the RYR protomer.