Trout cardiac troponin C (cTnC) contains differences in amino acid sequence that are partially responsible for the disparity in function between trout and mammalian hearts. Interestingly, the physiological temperature and amino acid sequence of cTnC from Xenopus laevis are intermediate between trout and mammals. As a result, the study of Xenopus cTnC may provide insight into the influence of physiological temperature on the evolution of cardiac function. Circular dichroism demonstrated that the melting temperature of the rat troponin complex was 6°C higher than one containing trout components, with Xenopus falling between. Ca2+-binding measurements indicated that Xenopus and trout troponin components increase the Ca2+-affinity of the troponin complex. The Ca2+ off-rates (koff) of the complexes containing trout and Xenopus components were also lower than the rat complex. This suggests that the Ca2+-affinity and thermal stability of the troponin complex are both related to the physiological temperature of the species.