Acclimation to cold (4°C) increases basal heart rate in rainbow trout (Oncorhynchus mykiss). The aim of this study was to test the hypothesis that thermal acclimation modifies the pacemaker mechanism of the trout heart. To this end, pacemaker potentials were recorded in intact sinoatrial tissue and enzymatically isolated pacemaker cells of trout acclimated at 4°C (cold-acclimated, c.a.) or 18°C (warm-acclimated, w.a.). With electrophysiological recordings the primary pacemaker was located at the base of the sinoatrial valve, and histological examination indicated a morphologically distinct ring of tissue comprising myocytes and neural elements in this location. Intrinsic beating rate (beats/min) of the pacemaker was higher in c.a. (50 ± 1) than w.a. (42 ± 3) (p < 0.05) trout at 11°C, and a similar difference was found in enzymatically isolated pacemaker cells (46 ± 2 vs. 39 ± 2) (p < 0.05). Inhibition of sarcoplasmic reticulum Ca release with 10 mM ryanodine and 1 mM thapsigargin did not effect heart rate in either w.a. or c.a. trout at 11°C. Half-maximal blockade of the delayed rectifier K⁺ current (I_Kr) with 0.1 mM E-4031 at 11°C reduced heart rate more in w.a. (from 45 ± 1 to 24 ± 5) than c.a. trout (from 56 ± 3 to 48 ± 2) (p < 0.05). Collectively, these findings indicate that thermal acclimation modifies the primary pacemaker mechanism of the trout heart, and suggest that temperature-induced differences in AP discharge frequency are partly due to lower density of the I_Kr in the w.a. trout, while contribution of sarcoplasmic reticulum Ca release to thermal compensation of heart rate is negligible.