While many reports have shown that Ca²⁺ alone causes ryanodine binding to the heavy fraction of the sarcoplasmic reticulum (HFSR), our results demonstrate that caffeine or β, γ-methylene adenosine triphosphate (AMPOPCP) in addition to Ca²⁺ is necessary for ryanodine binding, although Ca²⁺ is indispensable for it. While clarifying the reasons for this discrepancy, we found that a high osmolarity of the reaction medium, but not ionic strength, is a crucial factor. In a hypertonic solution containing 1M NaC1, Ca²⁺ alone causes a sizable extent of ryanodine binding. Caffeine and AMPOPCP independently stimulate it, unlike the case of 0.17M KC1 (or NaC1) medium, in which they show a potentiating interaction. Ryanodine binding in the hypertonic solution was markedly enhanced not only as to the binding rate but also the extent. The Scatchard plot was linear, indicating a single class of homogeneous binding sites. The maximum number of binding sites as well as the affinity was also increased in 1M NaCI-medium. The presence of AMPOPCP and/or caffeine did not affect the magnitudes of them so much, especially that of the affinity, in the hypertonic medium, as in the isotonic medium. The Ca²⁺ dependence of ryanodine binding in the stimulatory range was similar to that in 0.17M KCl- (or NaC1-) medium. However, the very weakinhibition at high Ca²⁺ concentrations is in striking contrast to ryanodine binding in the isotonic medium. The stimulation due to a high osmolarity is distinct, as to the mechanism, from that due to AMPOPCP, caffeine, or temperature. The dissociation of [³H] ryanodine bound was also examined under various experimental conditions.