The Ca2+-dependent ryanodine binding site of rabbit cardiac sarcoplasmic reticulum is solubilized by treatment with 20 mM CHAPS detergent and 1 M NaCl for 30 min at 0 degrees C. Ca2+ added at 5 microM enhances binding, at 0.5 mM increases both the affinity and number of [3H]ryanodine binding sites, while at 10 mM only the number of binding sites is increased. Mg2+ up to 1 mM does not significantly affect [3H]ryanodine binding. Radioligand binding is strongly enhanced by all alkali metal chlorides except LiCl. NaCl increases the rate of association of the ligand and the affinity of the binding site but does not influence the dissociation. NaCl and CaCl2 enhance the thermal stability of the [3H]ryanodine-binding protein. Thiol groups are essential for [3H]ryanodine binding. Ruthenium red and Cd2+ inhibit binding, while theophylline is stimulatory at low (micromolar) Ca2+ concentrations by a mechanism other than phosphodiesterase inhibition. Gel permeation chromatography establishes that the ryanodine binding protein is localized only in the high molecular mass fraction (greater than 669 kDa). Polyacrylamide gel electrophoresis of the proteins following treatment with SDS and 2-mercaptoethanol indicates that more than 90% are of low molecular mass (34-70 kDa) and that two stain blue with Stains-all as expected of Ca2+-binding proteins.