The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.