Because surprising similarities exist between the cardiac effects of amiodarone and those observed during hypothyroidism, we tested three different mechanisms of action in rats to determine whether amiodarone might act by inducing a tissular hypothyroidism: (a) a decrease in thyroid secretion, (b) an inhibiting effect on the conversion of thyroxine (T4) to 3,5,3′ triiodothyronine (T3), and (c) a direct antagonistic effect on the cellular action of T3. Five groups of rats, treated orally for 7 or 13 days, were studied: I, control (0.5 ml saline for 7 or 13 days, n = 14); II, amiodarone (50 mg/kg for 7 days, n = 5); III, iopanoic acid (100 mg/kg for 13 days, n = 7); IV, control + T3 (0.5 ml saline for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5); V, amiodarone + T3 (amiodarone 50 mg/kg for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5). Cardiac β-adrenoceptor density (CBARD) and heart rate (HR) were the two endpoint parameters investigated. Thyroid status was evaluated by serum thyrotropin (TSH), T4, T3, rT3 concentrations and liver type I 5′-deiodinase (5′D-I) activity. Amiodarone (group II) decreased CBARD (−22%, p < 0.05) without altering thyroid secretion and T3 serum level, whereas 5'D-I was strongly inhibited (−90%, p < 0.01). Iopanoic acid had no effect on CBARD and HR, but deeply inhibited 5'D-I. The increase in CBARD (+ 89%, p < 0.05) and in HR (+141 beats/min, p < 0.05) induced by T3 treatment (group IV) was partially inhibited in group V by amiodarone ( + 46% CBARD, +120 beats/min HR, p < 0.05 vs. group IV) in a noncompetitive way. CBARD was also linearly related to T3 serum levels, and combined results of groups IV and V suggest that the effects of amiodarone on CBARD are T3-dependent and that amiodarone decreases HR by reducing CBARD, still in a linear way. We conclude that the lowering effect of amiodarone on CBARD in euthyroid rat is clearly related to an antagonistic effect on T3 at the cardiac cellular level. An inhibition of thyroid hormone secretion or a decrease in T4 to T3 conversion can be ruled out.