Thyroxine (T4) and triiodothyronine (T3) were measured in thyroid glands obtained from patients dying of nonthyroidal illnesses and from patients undergoing surgery for Graves' disease, follicular adenoma, colloid goiter, or chronic thyroiditis. The thyroid glands (saline extract, homogenate, or thyroglobulin [Tg]) were hydrolysed with Pronase under anaerobic conditions in the presence of 0.05m methylmercaptoimidazole and hydrolysates extracted with butanol-ethanol. Aliquots of the extracts were evaporated and analyzed for T4 content by competitive-protein binding and/or by radioimmunoassay and for T3 content by radioimmunoassay. The mean (± sem) T4 content (μg/g tissue) in 25 glands from euthyroid subjects was 172.2 ± 22.1 and the T4/T3 ratio 19.5 ± 1.97. Eight thyroid homogenates were processed by thin layer chromatography to separate T4 from T3 followed by iodometry. The T4/T3 ratio of 7.0 ± 1.0 so obtained was significantly lower than that (19.9 ± 3.3) obtained in these glands by specific radioimmunoassay methods. These data suggest that lower values of T4/T3 ratio (2.0–7.6) reported previously in normal thyroid glands may have resulted in part, at least, from methodologic problems of earlier methods. The T4 content of 18.6 ± 1.99 μg/g tissue and the T4/T3 ratio of 2.87 ± 0.78 in thyroid glands of patients with Graves' disease was significantly lower than that in euthyroid subjects. The T4 content and the T4/T3 ratio were comparable to normal in patients with follicular adenoma. In glands with chronic thyroiditis the T4 content was subnormal whereas the T4/T3 ratio was significantly elevated. However, an unusual white precipitate had formed in the Pronase hydrolysates of these glands which limits the interpretation of the high T4/T3 ratio.