Potassium currents are plastic entities which modify electrical activity of the heart in various physiological conditions including chronic thermal stress. We examined molecular basis of the inward rectifier K current (I_K1) in rainbow trout acclimated to cold (4°C, c.a.) and warm (18°C, w.a.) temperature. Kir2.1 and Kir2.2 transcripts were expressed in atrium and ventricle of the trout heart, Kir2.1 being the major component in both cardiac chambers. The relative expression of Kir2.2 was, however, higher (P<0.05) in atrium than ventricle. The density of ventricular I_K1 was about 25% larger (P<0.05) in w.a. than c.a. trout. Furthermore, the I_K1 of the w.a. trout was ten times more sensitive to Ba²⁺ (IC₅₀, 0.18 ± 0.42 µM) than the I_K1 of the c.a. trout (1.17 ± 0.44 µM) (P<0.05) and opening kinetics of single Kir2 channels was slower in w.a. than c.a. trout (P<0.05). When expressed in COS-1 cells, the homomeric Kir2.2 channels demonstrated higher Ba²⁺ sensitivity (2.88 ± 0.42 µM) than Kir2.1 channels (24.99 ± 7.40 µM) (P<0.05). Based on different Ba²⁺ sensitivities of omKir2.1 and omKir2.2 channels, it is concluded that warm-acclimation increases either number or activity of the omKir2.2 channels in trout ventricular myocytes. The functional changes in I_K1 are independent of omKir2 transcript levels which remained unaltered by thermal acclimation. Collectively, these findings suggest that thermal acclimation modifies functional properties and subunit composition of the trout Kir2 channels, which may be needed for regulation of cardiac excitability at variable temperatures.