The intracellular Na⁺-concentration, [Na⁺]_i, modulates excitation-contraction (e-c) coupling of cardiac myocytes via the Na⁺/Ca²⁺-exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole-cell patch-clamp studies have shown that Ca²⁺-influx via reverse-mode NCX contributes significantly to contraction when [Na⁺]_i is 16 mM but not 10 mM. However, physiological [Na⁺]_i has never been measured. We recorded [Na⁺]_i using the fluorescent indicator SBFI in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na⁺]_i at rest and during increases in contraction frequency across three temperatures which span those the trout experience in nature (7, 14 and 21 °C). Surprisingly, we found that [Na⁺]_i was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na⁺]_i in resting cells. Thus, we report a resting in vivo [Na⁺]_i of 13.4 mM for rainbow trout cardiomyocytes. [Na⁺]_i increased from rest with increases in contraction frequency by 3.2 %, 4.7 %, and 6.5 % at 0.2, 0.5 and 0.8 Hz, respectively. This corresponds to an increase of 0.4 mM, 0.6 mM and 0.9 mM at 0.2, 0.5 and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na⁺]_i. Our results provide the first measurement of [Na⁺]_i in rainbow trout cardiomyocytes. This surprisingly high [Na⁺]_i is likely to result in physiologically significant Ca²⁺-influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca²⁺-source will decrease with the action potential duration as temperature and contraction frequency increases.