Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+)-release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca(2+) is unclear. Here, we characterized the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1±1.8 versus 30.3±2.4ms) whereas with isoproterenol (1µM), inactivation was significantly faster with EGTA (11.6±1.7 versus 27.3±1.6ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9±6.1 versus 68.0±8.7ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+), whilst with isoproterenol, inactivation was Ca(2+)-dependent (~65%) and highly reliant on SR Ca(2+) (~46%). Thus, SR Ca(2+) is not released in basal conditions and ICa is the main trigger of contraction whereas during a stress response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not attributed to differences in SR Ca(2+) load because caffeine-induced transients were not different in both conditions. Therefore, Ca(2+) 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.