The Ca2+ sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca2+ titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 °C and F27W mutant at 30 °C) and fluorescence (F27W mutant, at 7 and 30 °C) spectroscopies. Using NMR spectroscopy, we detected Ca2+ binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca2+ during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 °C. Despite the low Ca2+ affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.