Temperature-induced changes in cardiac output (Q) in fish are largely dependent on thermal modulation of heart rate (fH) and at high temperatures Q collapses due to heat-dependent depression of f_H. This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of f_H in vivo. To this end temperature-dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, f_PM), spontaneous beating rate of isolated sinoatrial preparations (f_SA) and in vivo heart rate (f_H) of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, f_PM steadily increased due to the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (T_BP) of 24.0±0.37°C where the electrical activity abruptly ceased. The maximum f_PM at T_BP was much higher (193±21.0 beats per minute (bpm)) than the peak f_SA (94.3±6.0 bpm at 24.1°C) or peak f_H (76.7±2.4 at 15.7±0.82°C) (P<0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit f_H at high temperatures in the brown trout in vivo.