Cardiomyocyte contraction depends on rapid changes in intracellular Ca²⁺. In mammals, Ca²⁺ influx as L-type Ca²⁺ current (I_Ca) triggers the release of Ca²⁺ from sarcoplasmic reticulum (SR) and Ca²⁺-induced Ca²⁺ release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca²⁺ is unclear. Here, we characterized the role of I_Ca to trigger SR Ca²⁺ release in rainbow trout ventricular myocytes using I_Ca regulation by Ca²⁺ as an index of CICR. I_Ca was recorded with a slow (EGTA) or fast (BAPTA) Ca²⁺ chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of I_Ca inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of I_Ca inactivation was due to Ca²⁺, while with isoproterenol, inactivation was Ca²⁺-dependent (∼65%) and highly reliant on SR Ca²⁺ (∼46%). Thus, SR Ca²⁺ is not released in basal conditions, and I_Ca is the main trigger of contraction, whereas during a stress response, SR Ca²⁺ is an important source of cytosolic Ca²⁺. This was not attributed to differences in SR Ca²⁺ load because caffeine-induced transients were not different in both conditions. Therefore, Ca²⁺ stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.