Na+/I- symporter (NIS) mediates iodide (I-) uptake in the thyroid gland, the first and rate-limiting step in the biosynthesis of the thyroid hormones. The expression and function of NIS in thyroid cells is mainly regulated by TSH and by the intracellular concentration of I-. High doses of I- for 1 or 2 days inhibit the synthesis of thyroid hormones, a process known as the Wolff-Chaikoff effect. The cellular mechanisms responsible for this physiological response are mediated in part by the inhibition of I- uptake through a reduction of NIS expression. Here we show that inhibition of I- uptake occurs as early as 2 hours or 5 hours after exposure to excess I- in FRTL-5 cells and the rat thyroid gland, respectively. Inhibition of I- uptake was not due to reduced NIS expression or altered localization in thyroid cells. We observed that incubation of FRTL-5 cells with excess I- for 2 hours increased H2O2 generation. Furthermore, the inhibitory effect of excess I- on NIS-mediated I- transport could be recapitulated by H2O2 and reverted by reactive derived oxygen species scavengers. The data shown here support the notion that excess I- inhibits NIS at the cell surface at early times by means of a posttranslational mechanism that involves reactive derived oxygen species.