Iodine incorporation into thyroglobulin is dependent on the activities of both thyroperoxidase (TPO) and thyroid dual oxidase 2 (DuOx2). Although TPO expression is decreased in some thyroid nodular lesions, DuOx1 and 2 mRNA expressions are maintained, but DuOx H 2 O 2 -generating activity has never been evaluated in such tumors. Our goal was to determine DuOx activity in hypofunctioning lesions of the thyroid. We evaluated H 2 O 2 generation by DuOx in 12 paranodular to cold nodule samples, 17 non-toxic multinodular goiters (MNG; 33 samples), 3 papillary carcinomas (PC; 4 samples), 3 follicular carcinomas (FC; 4 samples), and 10 follicular adenomas. DuOx activity was detected in all paranodular tissues (121G23 nmol H 2 O 2 /h per mg protein), but was undetectable (!1 nmol H 2 O 2 generated) in all PC, two out of four FC samples and seven out of ten adenomas. In 11 MNG at least two different areas of the goiter have been evaluated, and in 5 of these goiters one of the samples had DuOx activity below the limit of detection. The coefficient of variation in MNG samples ranged from 11.3 to 57.2%. Interestingly, in all the adenomas studied, TPO activity (486G142 U/g protein, nZ8) was well within the range found in paranodular tissues (414G116 U/g protein, nZ3). We found a significant negative correlation between DuOx and TPO activities, suggesting that these enzymes are regulated in opposite directions, at least in thyroid tumors.