Nitric Oxide (NO) is a free radical that mediates cell communication and signal transduction in numerous physiological and pathological processes. The main target of NO is the soluble guanylyl cyclase which catalyzes the formation of 3´5´-cyclic guanosine monophosphate (cGMP). CyclicGMP activates a variety of effectors such as protein kinase cGMP-dependent (PKG). Several effects of NO on thyroid function have been reported. The three isoforms (I-III) of NO synthase (NOS), the NO generating enzyme, have been demonstrated in thyroid tissue. This work aimed to study the role of endogenous NO on thyroid hormone biosynthesis in the rat thyroid cell line FRTL-5. The treatment of cells with the NOS inhibitor nitro-L-arginine-methyl-ester (L-NAME) (1 mM) induced an increase of TSH-stimulated (200-1000 µUI/ml) iodide uptake at 24-72h with a maximum at 48h. A significant increase of TSH-stimulated iodide uptake was also obtained when cells were incubated for 48 h with 5 µM KT-5823, a PKG inhibitor. Preliminary observations indicated that L-NAME treatment increased TSH-induced Tg mRNA expression (Northern-blot). Production of NO (nitrite accumulation) was not detected under 200-1000 µUI/ml TSH stimulation for 24-72h. However, an increase of NOS III mRNA expression (in situ hibridization) was induced by TSH. Values were (mean±SEM, cpm/well, 48h): Basal: 498±57, 200µUI/ml TSH: 1203±231** (**p< 0.01, n=6, Student t test).  In conclusion, this findings evidence an inhibitory effect of endogenous NO on iodide uptake probably involving a cGMP/PKG-mediation. A role of the NO/cGMP pathway as a negative signal in the regulation of TSH-stimulated thyroid hormone biosynthesis is supported.