A thyroid hormone-binding substance (TBGw) similar to serum T4-binding globulin (TBG) has been identified in the whey fraction of human breast milk. TBGw coeluted with serum TBG, as determined by Bio-Gel P-100 chromatography, and has an isoelectric point of 4.2-4.8, similar to that of serum TBG. The affinity constant of TBGw for T4 was similar to that of serum TBG(Ka, 1.54 +/- 0.38 X 10(9) M-1). Marked inhibition of TBGw binding of [125I]T4 was achieved by the addition of 1.5 X 10(-3) M 8-anilino-1-naphthalene-sulfonic acid. An albumin-like low affinity site (Ka, greater than 10(7) M-1) was also found. RIA of whey concentrates serially diluted in TBG-depleted serum indicated nearly identical binding curves for TBGw and TBG, with slopes of 2.33 (n = 15; r = 0.947) and 2.54 (n = 7; r = 0.996), respectively. Using a specific TBG RIA, a TBGw concentration of 0.29 +/- 0.08 microgram/ml (mean +/- SD) in breast milk (n = 26) was determined. Paired serum and whey specimens from individuals between 6 and 20 weeks of lactation were analyzed by TBG RIA; in these individuals, mean serum TBG concentrations were 25.0 +/- 3.4 micrograms/ml (n = 7); corresponding TBGw levels were 0.26 +/- 0.08 micrograms/ml (n = 7), i.e. approximately 1% of serum levels. Nonetheless, linear regression analysis of the data revealed no significant correlation between serum and whey TBG concentrations in these individuals.