The serum T3 assay has been regarded as the most sensitive single test for hyperthyroidism although impaired conversion of T4 to T3 in non-thyroidal illness (NTI) might decrease its diagnostic sensitivity. The present report gives experience from the T3 assay in middle-aged females under conditions similar to those in a general health survey. The assays were performed during two periods with an interval of six years. In 1974-75 we studied a representative sample (n = 1283) of women of ages 44, 52, 56, 60 and 66 years in Göteborg, Sweden. Individuals with serum T3 concentration greater than mean + 2.5 SD were selected for a follow-up study (n = 21). Of 16 individuals with no previous thyroid disease and no present treatment with thyroid hormones or oestrogens, 14 were subjected to a TRH-stimulation test giving a normal TSH response in 10 cases having T3 concentrations up to mean + 3.5 SD. Four women with serum T3 concentration greater than or equal to mean + 3.5 SD had previously unrecognized autonomous function thyroid function, of whom two developed hyperthyroidism after two years. The original population sample was reinvestigated after six years in 1980-81 (n = 1138) together with an additional sample of women giving a total sample of 1422 women of ages 26, 38, 50, 58, 62, 66 and 72 years. Of the females studied in 1974-75 eight had developed hyperthyroidism between the two studies; three of these had raised serum T3 at the investigation in 1974-75. No case of hyperthyroidism had been missed by the T3 assay in the 1974-75 study. Of individuals with serum T3 greater than or equal to mean + 2.5 SD selected for a follow-up (n = 29) at least five were found to have previously unrecognized thyroid autonomy. We found a raised serum T3 to be associated with hyperthyroid (n = 2) and euthyroid Graves' disease, autonomously functioning thyroid adenoma(s), possible painless subacute thyroiditis, possible thyrotoxicosis factitia, diminished thyroid reserve and thyroid substitution therapy.