The cardiac Na⁺/Ca²⁺ exchanger (NCX), an important regulator of cytosolic Ca²⁺ concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7°C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents (I_NCX) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. The I_NCX for both isoforms exhibited Na⁺-dependent inactivation and Ca²⁺-dependent positive regulation. The I_NCX of NCX1.1 exhibited typical mammalian temperature sensitivities with Q₁₀ values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I_NCX of NCX-TR1.0 was relatively temperature insensitive with Q₁₀ values of 1.2 and 1.1 for peak and steady-state currents, respectively. I_NCX current decay was fit with a single exponential, and the resultant rate constant of inactivation () was determined as a function of temperature. As expected,  decreased monotonically with temperature for both isoforms. Although  was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on  was not different between the two isoforms. These data suggest that the disparities in I_NCX temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after -chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I_NCX temperature dependence between the two isoforms are not due to potential disparities in either the I_NCX regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.