A radioimmunoassay for the estimation of 3,5-diiodothyronine (3,5-T2) in human urine has been established. The urinary excretion of both glucuronide and sulfate conjugates of 3,5-T2 were estimated after enzymatic deconjugation. In 19 healthy controls the median excretion of unconjugated 3,5-T2 was 276 pmol/d, whereas the median excretion of glucuronidated and sulfated 3,5-T2 in 7 healthy subjects was 448 and 451 pmol/d, respectively. The median excretion of 154 pmol/d in 9 hypothyroid patients did not differ from that found in controls. In contrast 12 patients with hyperthyroidism had an enhanced excretion, 1312 pmol/d (P less than 0.01). Compared with previous data on the daily degradation of 3,5-T2, it is concluded that approximately one-sixth of degradated 3,5-T2 is excreted in the urine.