The cardiac Na⁺-Ca²⁺ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild type trout and canine NCX cDNA, and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14 and 30°C using the giant excised patch technique. Using this approach, the differential temperature dependence of NCX was found to reside within the N-terminal region of the molecule. Specifically, we found that approximately 75% of the differential energy of activation is at Na⁺-Ca²⁺ exchange ributable to sequence differences in the region that includes the first four transmembrane segments and the remainder to transmembrane segment five and the exchanger inhibitory peptide (XIP) site.