There is little agreement among published reports of the radioimmunoassayable thyroid hormone content of breast milk, likely due to wide variations in methodology applied. In order to achieve a higher degree of specificity in the determination of T<sub>3</sub> and T<sub>4</sub> concentrations in breast milk, samples were ethanol-extracted and then chromatographed on an LH-20 column. Using this method, all T<sub>3</sub> and T<sub>4</sub> RIA activity eluted with the void volume. Following pancreatin digestion and subsequent extraction of whole milk samples, void volume T<sub>3</sub> RIA activity decreased, and T<sub>3</sub> co-eluted primarily with a standard preparation of T<sub>3 </sub>or <sup>I25</sup>I-T<sub>3</sub>, at a concentration of 275 ± 132 ng/dl (mean ± SD) (n = 9). In contrast, the elution volume of T<sub>4</sub> RIA activity appeared unaffected by pancreatin. These data indicate that immunoreactive T<sub>3</sub> and T<sub>4</sub> are differentially bound to a thyroid hormone ‘binding’ substance present in breast milk. They further support the hypothesis that thyroid hormone sufficient to supplement the thyroid economy of the thyroid-deficient suckling infant is present in human breast milk.