A radioimmunoassay for the determination of thyroxine (T4) and triiodothyronine (T3) in urine was developed. The extraction of a sample, the incubations with T3-und subsequently T4-antibody and the elutions of the respective bound fractions are performed all on the same Sephadex column. This principle can be applied to as many as 120 simultaneous determinations. The interassay coefficients of variation were 20.1% for T3 and 10.6% for T4, respectively. The recovery of standards added to urine was 107 +/- 8% (mean +/- SD) for T3 and 102 +/- 8% for T4. The 24 h collections of urine from healthy controls contained 1.70 +/- 0.40 mug T3 and 1.44 +/- 0.51 mug T4 (mean +/- SD, N=20). Three hourly fractions of the urinary excretion collected from 6 a.m. through 9 p.m. showed a significant (p less than 0.0005) elevation of the T4 excretion as compared to the night fraction, whereas an increased T3 excretion was only observed from 6-9 P.M. The T4 excretion was reduced in primary hypothyroidism (0.48 +/- 0.47 mug per 24 h, p less than 0.0005), whereas the T3 excretion was not that markedly reduced (1.30 +/-0.80 mug/d), pointing to the known residual T3 secretion. A reduced T4 excretion (0.85 +/- 0.34 mug/d, p less than 0.00005) contrasted with the normal T3 excretion in patients with endemic goiter, indicating the known preferential T3 secretion in iodine deficiency states. T3 thyrotoxicosis was accompanied by an elevated T3 excretion with normal urinary T4. Normal excretion of T4 and T3 were observed in patients with persistantly suppressed serum TSH levels after successful treatment for hyperthyroidism.