Desethylamiodarone (DEA) is the major metabolite of amiodarone and has similar electrophysiologic effects with prolongation of the repolarization that is reversed by thyroid hormone (T3). Some of the electrophysiologic effects are probably due to antagonism of T3 at the receptor level. Such effects of T3 are mediated by modulation of gene transcription. The aim of this study was to investigate whether cycloheximide (Cy), an inhibitor of protein synthesis, and actinomycin D (ActD), a RNA-synthesis inhibitor, block DEA-induced prolongation of the repolarization and whether DEA takes part in the autoregulation of the nuclear thyroid hormone-receptor subtypes (ThR). Corrected monophasic action potentials (MAPc) and QTc were measured in Langendorff-perfused guinea pig hearts for 1 h. The hearts were continuously perfused with (a) vehicle, (b) 7.5 μM Cy, (c) 5 μM DEA, (d) 5 μM DEA + 7.5 μM Cy, (e) 1 μM T3, (f) 5 μM DEA + 1 μM T3, (g) 1.5 μM ActD, and (h) ActD + DEA. A potassium channel blocker with class III antiarrhythmic effects, 0.5 μM almokalant, was used as a control, separately and together with Cy. Western blot analysis for the ThR subtypes α, β1, and β2 was performed on vehicle- and DEA-treated hearts. DEA increased MAPc by 19% (p < 0.0005) and QTc by 18% (p < 0.0005). There was no effect on MAPc or QTc when Cy, ActD, or T3 was added with DEA. Almokalant increased MAPc by 14% (p < 0.005) and QTc by 13% (p < 0.0005). When Cy was present, almokalant still induced a similar prolongation of MAPc by 14% (p < 0.005) and QTc by 17% (p < 0.0005). Western blot analysis revealed no change in the expression of the ThR protein. In conclusion, the prolongation of the cardiac repolarization by DEA, but not almokalant, can be totally blocked by Cy and ActD. This indicates that the class III action of DEA is at least in part dependent on transcription rather than a direct effect on cell-membrane channels or receptors. The action of DEA could be reversed by T3, indicating an antagonism between DEA and T3. These results suggest a new antiarrhythmic mechanism dependent on gene expression.