We previously reported that two sisters with juvenile hypothyroidism due to Hashimoto's thyroiditis (Case 1: 13 years old, Case 2: 10 years old) had antibodies against thyroid hormones. Treatment was started with 12.5 micrograms of L-T4 per day in Jan. 1980. The doses were gradually increased, and after 1 year of treatment, both patients were clinically euthyroid on 100 micrograms of L-T4 per day, and the heights of Case 1 and of Case 2 had increased by 9 cm and by 10 cm, respectively. Serum TSH levels were decreased from 1088 microU/ml to 1.7 microU/ml in Case 1, and from 1300 microU/ml to 2.1 microU/ml in Case 2. The titers of antithyroglobulin antibodies as measured by solid phase RIA decreased in both patients after the treatment. The bindings of 125I-T3 and of 125I-T4 to sera in the presence of 8-anilino-1-naphthalene sulfonic acid to block binding to TBG (non-treated sera) were markedly higher in the two patients before therapy than those in ten normal controls (11.8% and 52.3% in Case 1, 46.8% and 21.5% in Case 2, and 5.9 +/- 0.6% and 4.1 +/- 0.5% (mean +/- SD) in the controls, respectively). After the 1 year treatment, the bindings of 125I-T3 and 125I-T4 decreased to normal levels in Case 1 (5.8% and 3.8%, respectively). In Case 2, the 125I-T4 binding decreased to the normal level (4.7%), whereas the 125I-T3 binding decreased but still remained above the normal level (10.6%). In order to exclude the interference of endogenous and/or therapeutic thyroid hormones with the binding of labelled hormones to sera, the sera were treated with dextran-coated charcoal at pH3.0 (acid-treated sera). The bindings of 125I-T3 and 125I-T4 to acid-treated sera were clearly higher in both patients before therapy than those in ten normal controls (16.9% and 60.7% in Case 1, 75.0% and 46.4% in Case 2, and 6.9 +/- 0.7% and 6.8 +/- 0.7% (mean +/- SD) in the controls, respectively), and these values were compatible with those from non-treated sera. After the 1 year treatment, however, the results of acid-treated sera were different from those of non-treated sera. That is, the bindings of 125I-T3 and of 125I-T4 to acid-treated sera from both patients decreased but remained above normal levels (9.3% and 26.5% in Case 1, and 29.4% and 22.5% in Case 2, respectively). These results indicate the presence of antibodies against thyroid hormones even in the euthyroid state during L-T4 treatment, and also the data obtained from the non-treated sera were affected by endogenous and/or therapeutic thyroid hormones. The importance of acid-charcoal treatment for the detection of anti-thyroid hormone antibodies during thyroid hormone administration was suggested. In the serum from another sister of the reported patients, we also found unusual T4-binding proteins which were only detected by the acid-charcoal treatment. The implications of decrement of anti-thyroglobulin antibodies and of anti-thyroid hormone antibodies were discussed.