The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4°C (c.a.) and 18°C (w.a.). With electrophysiological recordings the primary pacemaker was located at the base of sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in c.a. (46±6) than w.a. (38±3) (p<0.05) trout, and similar difference was seen in beating rate of isolated pacemaker cells (44±6 vs. 38±6) (p<0.05), thus supporting the hypothesis that thermal acclimation modifies intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 µM ryanodine and 1 µM thapsigargin did not affect heart rate in either w.a. or c.a. trout at 11°C, but reduced heart rate of w.a. trout from 74±2 to 42±6 beats/min (p<0.05) at 18°C. At 11°C, a half-maximal blockade of the delayed rectifier K current (IKr) with 0.1 µM E-4031 reduced heart rate more in w.a. (from 45±1 to 24±5) than c.a. (56±3 vs. 48±2) trout, while density of I_Kr was higher and duration of AP less in c.a. hearts (p>0.05). Collectively, these findings suggest that cold-induced increase in AP discharge frequency is at least partly due to higher density of the I_Kr in the c.a. trout, while contribution of SR Ca release to thermal compensation of heart rate is negligible.