Naturally occurring and synthetic plant flavonoids, such as EMD 21388, are potent inhibitors of thyroid hormone 5'-deiodinase (5'-D) in vitro, but not when given in vivo, since they are tightly bound by serum transthyretin (TTR). EMD 21388 also inhibits the binding of T4 to human, dog, and rat serum TTR in vitro and when administered to rats in vivo. In the present studies the administration of EMD 21388 inhibited the binding of T4 to TTR within 3 min, resulting in a decrease in the serum T4 concentration, an increase in the percentage of serum free T4 assessed by equilibrium dialysis, and an increase in the serum total free T4 concentration. Depending upon the dose of EMD 21388 employed, the serum total free T4 concentration was either elevated for at least 60 min or transiently elevated, returning to normal values by 60 min. Although the total serum T3 concentration was decreased and the percent free T3 increased, these changes were modest, and the serum free T3 concentrations remained normal after EMD 21388 administration. The transient elevations of serum free T4 concentrations 10 and 20 min after the administration of 0.3 mumol EMD 21388/100 g BW resulted in a significant decrease in the serum TSH concentration at 60 min. These observations strongly suggest that the serum free T4 concentration and not T4 bound to serum TTR is biologically available to the pituitary to regulate TSH secretion and/or synthesis. The administration of EMD 21388, which rapidly increases the serum free T4, but not the serum free T3, concentration, will now permit studies of the effect(s) of endogenously elevated serum free T4 concentrations, rather than those after the administration of pharmacological quantities of T3 and T4, on various aspects of the biosynthesis and release of pituitary TSH.