To investigate the properties of ryanodine binding sites of the bird skeletal SR vesicles, SDS PAGE, purification of RyR, and [3H]ryanodine binding study were carried out in the SR vesicles prepared from the chicken pectoral muscle. The chicken SR vesicles have two high molecular weight (HMW) protein bands as in eel SR vesicles on SDS PAGE. The HMW bands on SDS PAGE were found in the [3H] ryanodine peak fraction (Fr3-5) obtained from the purification step of the ryanodine receptor protein. Bmax and KD of the chicken [3H]ryanodine binding sites were 12.52 pmol/mg protein and 14.53 nM, respectively. Specific [3H]ryanodine binding was almost maximal at 50∼100 ՌM Ca2+, but was not increased by 5 mM AMP and not inhibited by high Ca2+. Binding was significantly inhibited by 20∼100 ՌM ruthenium red and 1 mM tetracaine, but slightly inhibited by Mg2+. From the above results, it is suggested that chicken SR vesicles have the ryanodine binding sites to which the binding of ryanodine is almost maximal at 50∼10 ՌM Ca2+, is significantly inhibited by ruthenium red and tetracaine, slightly inhibited by Mg2+, but not affected by AMP and not inhibited by high Ca2+.