Na /I symporter (NIS) transports iodide into thyrocytes, a fundamental step for thyroid hormone biosynthesis. Our aim was to evaluate NIS regulation in different status of goitrogenesis and its underlying mechanisms. Wistar rats were treated with methimazole (MMI) for 5 and 21 days, to achieve different status of goiter. We then evaluated the effect of MMI removal for 1 day (R1d), after 5 (R1d-5d) or 21 (R1d-21d) days of MMI treatment. MMI increased thyroid weight, iodide uptake and in vitro TPO activity in a time-dependent way. Although MMI removal evoked a rapid normalization of TPO activity in R1d-5d, it was still high in R1d-21d. On the other hand, iodide uptake was rapidly down-regulated in R1d-21d, but not in R1d-5d, suggesting that the increased TPO activity in R1d-21d led to increased intraglandular organified iodine (I-X), which is known to inhibit iodide uptake. Since TGFβ has been shown to mediate some effects of I-X, we evaluated TGFβ and TGFβ receptor mRNA levels, which were increased in R1d-21d. Moreover, it has been demonstrated that TGFβ stimulates NOX4. Accordingly, our data revealed increased NOX4 expression and H O generation in R1d-21d. Finally, we evaluated the effect of H O on NIS function and mRNA levels in PCCL3 thyroid cell line, which were reduced. Thus, the present study suggests that there is a relationship between the size of the goiter and NIS regulation and that the mechanism might involve I-X, TGFβ, NOX4 and increased ROS production.