Thyroid side-effects are common in patients treated for cardiac arrhythmias with amiodarone (AM). A major disturbance is inhibited thyroidal radioiodine uptake in AM-induced thyrotoxicosis (AIT), which makes ¹³¹I therapy ineffective. On the other hand, failure to escape from the Wolff-Chaikoff effect by down-regulation of the sodium/iodide symporter (NIS) is proposed to explain AM-induced hypothyroidism (AIH). However, previously no experimental studies on the possible mechanisms have been conducted. We therefore investigated the early effects of AM on thyroidal iodide transport using bicameral chamber cultures of primary pig thyrocytes that reproduce the three tissue compartments (epithelium, lumen and extrafollicular space) of the gland. AM dose-dependently (1-50 µM) inhibited the TSH-stimulated transepithelial (basal-to-apical) transport of ¹²⁵I^- by up to 90%. The inhibitory effect was noticed already after 8 h, and was further pronounced after 1-4 days depending on the AM concentration. The intracellularly accumulated ¹²⁵I^- was reduced by perchlorate but not AM, and quantitative real-time RT-PCR revealed no change in the NIS expression in AM-treated cells. Blocking of cyclic AMP degradation with IBMX or withdrawal of AM reversed AM-induced changes in electrolyte transport but were unable to recover the suppressed ¹²⁵I^- transport. The iodine-free AM analog dronedarone also inhibited ¹²⁵I^- transport to the same extent as AM. The findings indicate that AM blocks thyroidal iodide uptake by reducing the iodide permeability of the apical plasma membrane of the thyroid epithelial cells. The effect is iodine-independent, long-lasting, and does not involve impaired function of NIS or the TSH receptor/cyclic AMP signaling pathway.