Opening and closing of the cardiac ryanodine receptor (RyR) is coordinated by the free intracellular Ca²⁺ concentration, thus making the Ca²⁺ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4°C as indicated by Ry-sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca²⁺-dependent kinetics of [³H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [³H]ryanodine binding sites determined at 20°C was 1.54 times higher in rat than burbot heart (0.401 ± 0.039 and 0.264 ± 0.019 pmol mg^-1 protein, respectively) (P<0.02), while binding affinity (K_d) for [³H]ryanodine was similar (3.38 ± 0.63 and 4.38 ± 1.14 nM for rat and burbot, respectively) (P=0.47). The high-affinity [³H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca²⁺ concentration at both 20°C and 2°C, and did not differ between the two species. Half-maximal [³H]ryanodine binding occurred at 0.191 ± 0.027 µM and 0.164 ± 0.034 µM Ca²⁺ for rat and at 0.212 ± 0.035 µM and 0.188 ± 0.039 µM Ca²⁺ for burbot (P=0.65), at 2°C and 20°C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca²⁺-binding affinity at 20°C was 4.4- and 5.9-times lower, respectively, than in the burbot. At 20°C, the rate of [³H]ryanodine binding to the high-affinity binding site was similar in rat and burbot, but was drastically slowed in rat at 2°C. At 2°C [³H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10° and 20°C the rate of dissociation was 2-3 times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism where the closing of the Ca²⁺ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [³H]ryanodine binding in rat in comparison to burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.