Objective: We studied the intervention of nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF) in mediating responses to acetylcholine in thyroid arteries from euthyroid (E) and methimazole-treated (MT) patients. Design and methods: Branches of the superior thyroid artery were obtained from 19 E patients and 17 MT patients (euthyroid at the time of surgery) undergoing total thyroidectomy or hemithyroidectomy. Artery rings were suspended in organ baths for isometric recording of tension. Results and conclusions: Acetylcholine caused endothelium-dependent relaxation of greater magnitude in arteries from MT patients (pD2 7.68±0.19 in E and 8.17±0.26 in MT, P<0.05). The relaxation was unaffected by indomethacin and partially reduced by the NO synthase inhibitor L-NMMA. This reduction was higher in arteries from MT patients (50±6 %) as compared to E patients (36±6 %) (P<0.05). Inhibition of K+ channels using apamin combined with charybdotoxin or high K+ solution abolished the relaxation resistant to L-NMMA and indomethacin. The maximal contractions to noradrenaline (in percentage of the response to 100 mM KCl) were lower in MT than in E patients (57±10 and 96±8, respectively, P<0.05). The hyporesponsiveness to noradrenaline in arteries from MT patients was corrected by L-NMMA. The results indicate: (1) thyroid arteries from MT patients show increased relaxation to acethylcholine and decreased contraction to noradrenaline due to overproduction of NO; (2) EDHF plays a prominent role in acetylcholine-induced relaxation through activation of Ca2+-activated K+ channels; (3) the abnormal endothelium-dependent responses in arteries from MT patients are not corrected by medical treatment.