The ryanodine receptor (RYR)/Ca2+ release channel of avian cardiac muscle was localized by immunocytochemical techniques and biochemically characterized using isolated membrane and receptor protein fractions. Monoclonal antibody C3-33 raised against the canine cardiac RYR bound to the junctional sarcoplasmic reticulum of pigeon and finch hearts, both at peripheral couplings and at extended junctional sarcoplasmic reticulum (EJSR). Immunoblots of sarcoplasmic reticulum vesicles from pigeon and finch hearts showed this antibody recognized a single high molecular weight protein, which co-migrated with the canine M(r) 565,000 RYR/Ca2+ release channel polypeptide. The pigeon heart RYR bound [3H]ryanodine with high affinity in a Ca(2+)-dependent manner, comparable to the canine cardiac RYR. Purification of the pigeon RYR yielded a 30 S protein complex, which bound the maximum calculated amount of [3H]ryanodine ((440 +/- 60) pmol/mg protein), assuming one high affinity site/tetrameric 30 S RYR comprised of M(r) 565,000 polypeptides. Autoradiography of isolated finch cardiac myocytes indicated [3H]ryanodine binding throughout the cells. These results suggest that avian heart contains a single population of RYRs, and thereby support the hypothesis that avian EJSR contains functional calcium release channels which, because of the absence of transverse tubules, can be located micrometers away from the surface membrane in avian heart.