Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca²⁺ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) plays a major role during cardiac E-C (excitation-contraction) coupling, transporting Ca²⁺ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca²⁺ uptake in SR enriched ventricular vesicles, indicated that tunas were capable of sustaining a rate of Ca²⁺-uptake that was significantly higher than the mackerel. Among tunas, the cold tolerant bluefin had the highest rates of SR Ca²⁺ uptake and ATPase activity. The differences among Ca²⁺ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca²⁺ uptake and ATPase activities seen in bluefin ventricles compared to albacore, yellowfin and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.