NO is a free radical that mediates numerous physiological and pathological processes. NO mainly acts on soluble guanylyl cyclase with formation of 3´5´-cyclic guanosine monophosphate (cGMP) which activates a variety of effectors such as the protein kinase cGMP-dependent (PKG). The three isoforms (I-III) of the NO generating enzyme, NO synthase (NOS), have been demonstrated in thyroid tissue. This work aimed to study the action of thyrotropin (TSH) on the NO production and the role of the endogenous NO on functional thyroid parameters in the rat thyroid cell line FRTL-5. Stimulation with TSH (200-500 µUI/ml) for 24-48h increased NO production (¹⁴C-citrulline). An increment of NOS III mRNA expression (in situ hibridization) was induced by TSH at 48h. The presence of the NOS inhibitor, L-NAME (1mM), induced an increase of the TSH-stimulated iodide uptake and TG mRNA expression (Northern Blot) at 48h. A significant increment of TSH-stimulated iodide uptake was also obtained when cells were incubated for 48h. with the PKG inhibitor, KT-5823 (5µM). In conclusion, these results indicate that the NO production could be induced by TSH in the thyroid cell. A possible role of NO as a negative signal in thyroid hormonogenesis was evidenced.