A radioimmunoassay procedure to measure triiodothyronine (T3) in unextracted urine is described. One hundred microliter of T3 standards or samples were incubated with 100 microliter of T3 antiserum (1:40,000), 100 microliter of tracer 125I-T3 and 700 microliter of 0.05 M borate buffer, pH 8.6. Separation of free from bound antigen was achieved by dextran coated charcoal. The cross-reaction of L-T3 with L-T4 in this assay system was less than 0.2%. Dilution of high T3 urine was parallel to the standard curve. Recovery was 101 +/- 8%. Coefficients of variation were 3-8% within each assay and 13% between assays. Euthyroid subjects excreted 0.81 +/- 0.39 microgram (mean +/- S.D.) in 24 hr. Similar values were obtained in pregnant women, euthyroid patients with chronic thyroiditis, and a patient with thyroxine-binding globulin deficiency. Excretion of urinary T3 was high (7.48 +/- 3.32 microgram/24 h) in patients with hyperthyroidism, and low (0.14 +/- 0.15 microgram/24 hr) in patients with hypothyroidism. Urinary T3 excretion was almost undetectable in terminal renal failure. Positive correlations were found between urinary T3 and serum total T3 (r = 0.89) and especially serum free T3 (r - 0.97). A positive correlation was also found between urinary T3 and creatinine clearance (r = 0.63). Mean urinary clearance of T3(CT3=Urinary T3 excretion/serum free T3 was 166 ml/min and was significantly higher than their mean creatinine clearance (110 ml/min). The measurement of T3 in urine is reliable and easy to perform, and may allow a new approach to the understanding of thyroid hormone metabolism.