Serum 3,3',5'-triiodothyronine (rT3) was measured with a radioimmunoassay in unextracted serum. The assay was specific and reproducible. The coefficients of variation for 3 different sera known for high, normal, and low rT3 concentrations between assays were 4, 6, and 9% within assays 4, 9, and 7%, respectively. In euthyroid subjects 20 to 60 years old, rT3 was 450 +/- 200 pg/ml (mean +/- 2 SD.n = 83). Serum rT3 was found to be increased in hyperthyroidism (range: 762-2581 pg-ml; n = 11) but also in acute and chronic illness (up to 2400 pg/ml; n = 24) and in anorexia nervosa (536-1058 pg/ml; n = 7). In the latter two situations there was mostly an inverse change in serum 3,5,3'-triiodothyronine (T3) which was in the low normal range or decreased. These findings suggest a metabolic control of thyroxine deiodination. A low serum rT3 was found in 9 of 12 hypothyroid patients and in the serum of 1 chronically ill patient. Long-term treatment (1-7 years) with lithium carbonate slightly reduced serum rT3, although the changes were inside the normal range. Kidney function was not found to be necessary for its production as anephric patients had normal rT3 values. In addition, hemodialysis increased serum rT3, which is probably due to the heparin therapy.