To explore how the cardiac sarcoplasmic reticulum (SR) functions over a range of temperatures, we used whole-cell voltage clamp combined with rapid caffeine application to study SR Ca(2+) accumulation, release and steady-state content in atrial myocytes from rainbow trout. Myocytes were isolated from rainbow trout acclimated to 14 degrees C, and the effect of varying stimulation pulse number, frequency and experimental temperature (7 degrees C, 14 degrees C and 21 degrees C) on SR function was studied. To add physiological relevance, in addition to 200 ms square (SQ) voltage pulses, myocytes were stimulated with temperature-specific action potentials (AP) applied at relevant frequencies for each test temperature. We found that the SR accumulated Ca(2+) more rapidly and to a greater concentration (1043+/-189 micromol l(-1) Ca(2+), 1138+/-173 micromol l(-1) Ca(2+), and 1095+/-142 micromol l(-1) Ca(2+) at 7 degrees C, 14 degrees C and 21 degrees C, respectively) when stimulated with physiological AP waveforms at physiological frequencies compared with 200 ms SQ pulses at the same frequencies (664+/-180 micromol l(-1) Ca(2+), 474+/-75 micromol l(-1) Ca(2+) and 367+/-42 micromol l(-1) Ca(2+) at 7 degrees C, 14 degrees C and 21 degrees C, respectively). Also, and in contrast to 200 ms SQ pulse stimulation, temperature had little effect on steady-state SR Ca(2+) accumulation during AP stimulation. Furthermore, we observed SR-Ca(2+)-dependent inactivation of the L-type Ca(2+) channel current (I(Ca)) at 7 degrees C, 14 degrees C and 21 degrees C, providing additional evidence of maintained SR function in fish hearts over an acute range of temperatures. We conclude that the waveform of the AP may be critical in ensuring adequate SR Ca(2+) cycling during temperature change in rainbow trout in vivo.