The binding of [3H]ryanodine with cardiac sarcoplasmic reticulum vesicles depends on the calcium concentration. Binding in the absence of calcium appears to be non-specific because it shows no saturation up to 20 microM ryanodine. The apparent Km value for calcium varied between 2 and 0.8 microM when the ryanodine concentration varied between 10 and 265 nM. The Hill coefficient for the calcium dependence of [3H]ryanodine binding was near two. Scatchard analysis of ryanodine binding indicated a high-affinity site with a Bmax of 5.2 +/- 0.4 pmol/mg with a Kd of 6.8 +/- 0.1 nM. Preincubation under conditions in which the high-affinity sites were saturated did not result in stimulation of the calcium uptake rate indicative of closure of the calcium channel. Stimulation of calcium uptake rate occurred only at higher concentrations of ryanodine (apparent Km = 17 microM). This stimulation of the calcium uptake rate also required calcium in the submicromolar range. The data obtained support the hypothesis that ryanodine binding to the low-affinity site (Km about 17 microM) is responsible for closure of the calcium release channel and the subsequent increase in the calcium uptake rate of the sarcoplasmic reticulum. Because the number of ryanodine-binding sites is much less than the number of calcium transport pumps the channel is probably distinct from the pump.