The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from +5 to +18°C (heat stress) shortened AP duration from 2.8 ± 0.3 to 1.3 ± 0.1 s in intact ventricles. In 56% (18 out of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K⁺ channels and increased their single channel conductance from 37 ± 12 pS at +8°C to 51 ± 13 pS at +18°C (Q₁₀ = 1.38) (p < 0.01; n = 12). The ATP-sensitive K⁺ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at +8°C (K_1/2 = 1.35 mM) and +18°C (1.85 mM). Although acute heat stress induced ATP-sensitive K⁺ current (I_K,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 µM) sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K⁺ currents from the same myocytes by alternate recording under current clamp and voltage clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K⁺ current, I_K1. In ~15% of myocytes (4 out of 27), I_K,ATP-dependent shortening of AP followed the I_K1-induced AP shortening. Thus, heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on I_K1. I_K,ATP is induced only in response to prolonged temperature elevation or perhaps in the presence of additional stressors.