Sodium nitroprusside (SNP) spontaneously produces nitric oxide (NO). In many cell types, this activates the soluble form of the enzyme guanylyl cyclase (GC), resulting in the elevation of cGMP. We herein report the role of NO and cGMP on iodide uptake in primary cultures of calf thyroid cells. Iodide uptake is the limiting step in thyroid hormone biosynthesis and a typical functional parameter. The effect of SNP on this parameter was thus determined. In cells treated with TSH for 72 h, addition of 5 mM SNP for the last 2 h caused a significant inhibition on iodide uptake, with no change in cells not treated with TSH. This action was mimicked by an analogue of cGMP, 8Br-cGMP, and blocked by reduced hemoglobin, thus suggesting that it is mediated by the GC-cGMP pathway. SNP also inhibited the stimulation caused by forskolin or analogues of cAMP, indicating that the effect takes place in this pathway, which would be distal to cAMP generation. The accumulation of radioiodine by thyroid cells is a consequence of the balance between influx and efflux. The studies demonstrate that SNP does not affect iodide efflux, thus revealing that it inhibits the influx.