SUMMARYAcute temperature change can be cardioplegic to mammals, yet certain ectotherms maintain their cardiac scope over a wide temperature range. To better understand the acute effects of temperature on the ectothermic heart,we investigated the stimulus-induced change in intracellular Ca2+concentration ([Ca2+]i; cytosolic Ca2+transient) in isolated rainbow trout myocytes at 7°C, 14°C and 21°C. Myocytes were voltage-clamped and loaded with Fura-2 to measure the L-type Ca2+ channel current (ICa) and[Ca2+]i during physiological action potential (AP)pulses at frequencies that correspond to trout heart rates in vivo at 7°C, 14°C and 21°C. Additionally, [Ca2+]iand ICa were examined with square (SQ) pulses at slow (0.2 Hz) and physiologically relevant contraction frequencies. The amplitude of[Ca2+]i decreased with increasing temperature for both SQ and AP pulses, which may contribute to the well-known negative inotropic effect of warm temperature on contractile strength in trout hearts. With SQ pulses, [Ca2+]i decreased from 474±53 nmol l-1 at 7°C to 198±21 nmol l-1 at 21°C,while the decrease in [Ca2+]i with AP pulses was from 234±49 nmol l-1 to 79±12 nmol l-1,respectively. Sarcolemmal Ca2+ influx was increased slightly at cold temperatures with AP pulses (charge transfer was 0.27±0.04 pC pF-1, 0.19±0.03 pC pF-1 and 0.13±0.03 pC pF-1 at 7°C, 14°C and 21°C, respectively). At all temperatures, cells were better able to maintain diastolic Ca2+levels at physiological frequencies with AP pulses compared with 500 ms SQ pulses. We suggest that temperature-dependent modulation of the AP is important for cellular Ca2+ regulation during temperature and frequency change in rainbow trout heart.