To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca21 release from the sarcoplasmic reticulum, we characterized Ca21 sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35 C to 10 C, the normalized fluo-3 fluorescence of Ca21 sparks decreased monotonically, but the D[Ca21]i were relatively unchanged due to increased resting [Ca21]i. The time-to-peak of Ca21 sparks, which represents the RyR Ca21 release duration, was prolonged by 37% from 35 C to 10 C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8 C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca21 concentration. These results provided a basis for quantitative interpretation of intracellular Ca21 signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25 C led to the notion that Ca21 sparks measured at a "warm room" temperature are basically acceptable in elucidating mammalian heart function.