Fatty acid metabolites accumulate in the heart under pathophysiological conditions that affect -oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K⁺ currents that control cardiac refractoriness and excitability. Transient outward (I_to) and inward rectifier (I_K1) K⁺ currents were recorded by the whole cell voltage-clamp technique in rat ventricular myocytes, and the effects of two major fatty acid metabolites were examined: palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA). Palmitoylcarnitine (0.5-10 µM) caused a concentration-dependent decrease in I_to density in myocytes internally dialyzed with the amphiphile; 10 µM reduced mean I_to density at +60 mV by 62% compared with control (P < 0.05). In contrast, external palmitoylcarnitine at the same concentrations had no effect, nor did internal dialysis significantly alter I_K1. Dialysis with palmitoyl-CoA (1-10 µM) produced a smaller decrease in I_to density compared with that produced by palmitoylcarnitine; 10 µM reduced mean I_to density at +60 mV by 37% compared with control (P < 0.05). Both metabolites delayed recovery of I_to from inactivation but did not affect voltage-dependent properties. Moreover, the effects of palmitoylcarnitine were relatively specific, as neither palmitate (10 µM) nor carnitine (10 µM) alone significantly influenced I_to when added to the pipette solution. These data therefore suggest that amphiphilic fatty acid metabolites downregulate I_to channels by a mechanism confined to the cytoplasmic side of the membrane. This decrease in cardiac K⁺ channel activity may delay repolarization under pathophysiological conditions in which amphiphile accumulation is postulated to occur, such as diabetes mellitus or myocardial infarction.