We have studied the origin of tri-iodothyronine (T3) secreted by human and sheep thyrocytes in primary culture and also the expression of type-I thyroidal iodothyronine deiodinase (ID-I) in the thyroid and liver of man and various other animals. Inhibitors of ID-I reduced T3 secretion from human but not sheep thyrocytes. In contrast, inhibitors of de-novo thyroid hormone synthesis reduced both thyroxine (T4) and T3 production in sheep thyrocytes, but had no effect on the T3 secreted by human thyrocytes. Human thyrocytes did not produce T4 under the culture conditions used, although some endogenous T4 was present in the cells following their isolation. Although thyrotrophin (TSH) stimulated T3 production in both human and sheep thyrocytes, iodine in the form of potassium iodide was only essential for T3 and T4 production by the sheep cells. Although 125I from Na125I was incorporated into T3 and T4 in TSH-stimulated sheep thyrocytes, no 125I incorporation into T3 or T4 was detected in TSH-stimulated human thyrocytes. Using activity measurements and affinity labelling, ID-I was present in the livers of all species studied, but ID-I could not be detected in thyroid tissue from cattle, pigs, sheep, goats, rabbits, deer or llamas. In contrast, thyroid tissue from man, mice, guinea-pigs and rats had significant ID-I activity and expressed an affinity-labelled protein with a molecular mass of approximately 28·1 kDa on SDS-PAGE. These data show that under the culture conditions used, sheep thyrocytes produced T3 by de-novo synthesis, whilst human thyrocytes produced T3 by deiodination of endogenous T4. We conclude that thyroidal ID-I shows marked species difference in its expression and that, in those species which express the enzyme (man, mice, guinea-pigs and rats, in this study), it appears that it may make an important contribution to thyroidal T3 production. Journal of Endocrinology (1993) 136, 361–370