1. A simple method is described for estimating the rate of thyroxine dissociation from binding sites in plasma.2. At tracer concentrations in human plasma thyroxine dissociates at two distinct exponential rates. These are due to the two thyroxine-binding proteins present in human plasma.3. The t((1/2)) for thyroxine dissociation from thyroxine-binding globulin is 8.1 +/- 0.7 (S.E.) min at room temperature and 38.6 +/- 2.1 sec at 37 degrees C.4. The t((1/2)) for thyroxine dissociation from thyroxine-binding pre-albumin is 53 +/- 5.0 sec at room temperature and 7.9 +/- 0.3 sec at 37 degrees C.5. Dissociation of thyroxine from both proteins is a single exponential process, suggesting that each protein has only one type of specific binding site.6. These results indicate that the minute pool of free thyroxine in plasma is turning over about 100 times each second, with about two thirds of this flux being due to release and binding of the hormone by pre-albumin.