The whey fraction of human milk contains a thyroxine-binding protein analogous to serum thyroxine-binding globulin. To assess the method by which this binding protein appears in milk over the course of lactation, paired serum and milk specimens from 45 healthy, euthyroid women were assayed for thyroxine-binding globulin and whey thyroxine-binding globulin using a highly specific thyroxine-binding globulin-radioimmunoassay. Elevated serum levels (39.2 +/- 4.72 micrograms thyroxine-binding globulin/mL; mean + SD, N = 5) were found in samples drawn at less than two weeks post partum. These levels normalized rapidly over the next three weeks post partum (26.0 +/- 3.3 micrograms/mL, P less than .005, N = 5), and by five weeks, no further significant decreases were observed. As was the case for serum thyroxine-binding globulin, whey thyroxine-binding globulin was present in its highest concentrations (79.3 +/- 13.4 micrograms/dL, N = 7) in the colostrum and transitional milk. These levels are equivalent to approximately 2 to 3% of serum concentrations. In contrast to serum thyroxine-binding globulin levels, however, whey thyroxine-binding globulin declined logarithmically over a protracted 12-week period, and no further significant changes in whey thyroxine-binding globulin concentrations were observed up to 20 weeks post partum, at which time they were approximately equal to 1% of serum thyroxine-binding globulin levels. A strong linear correlation between serum and whey thyroxine-binding globulin was found within the first five weeks post partum (r2 = .95, P less than .0005, N = 14).(ABSTRACT TRUNCATED AT 250 WORDS)