[en] The present report describes a RIA for 3,5-diiodothyronine (3,5T₂) which uses inner ring-labeled 3,5-[¹²⁵I]T₂ as the ligand and has a lower limit of detectability of 0.5 ng/dl. Cross-reaction was 0.14% with T₃, less than 0.001% with T₄, 1.2% with 3,3',5-triiodothyroacetic acid, and 6.1% with 3,5-diiodothyroacetic acid. No cross-reaction was detectable for iodothyronines within their physiological ranges. Intraassay variation ranged from 2.2 to 7.8%, and interassay variation ranged from 12.7 to 14%. The mean (+-SE) serum 3.5T₂ concentration in 70 normal subjects was 4.3 +- 0.2 ng/dl. The mean (+-SE) 3.5T₂ in 14 hyperthyroid patients was increased to 18.4 +- 2.3 ng/dl (P < 0.001), and all but 1 patient had an elevated level. In 10 hypothyroid patients the mean (+-SE) 3,5T₂ level was decreased to 1.4 +- 0.3 ng/dl (P < 0.001). In 4 patients, levels overlapped with the normal range. In 4 hypothyroid subjects treated with L-T₁, 3,5T₂ levels were normal, suggesting that the majority of 3,5T₂ originates from extrathyroidal conversion from T₃. Studies in fasting obese subjects demonstrated that serum 3,5T₂ (mean +- SE) levels fell from 3.4 +- 0.3 to 2.5 +- 0.7 ng/dl during fasting. This fall was significant (P < 0.001) and in parallel with the fall in T₃ levels of 182 +- 20 to 126 +- 12 ng/dl. In fasting subjects given 100 μg oral L-T₃/day T₃ levels rose from 138 +- 11 to 362 +- 26 ng/dl. 3,5T₂ levels (corrected for cross-reaction and for contamination of oral T₃ with 3,5T₂) rose from 2.2 +- 0.7 to 6.4 +- 1.0 ng/dl. In fasting subjects given 25 μg oral L-T₃/day, T₃ levels fell from 165 +- 5.1 to 139 +- 6.9 ng/dl. Corrected 3,5T₂ levels changed from 3.7 +- 0.4 to 2.5 +- 0.3 ng/dl. Neither change were significant