The binding characteristics of human TBG, TBPA and TBA of T4 and T3 were studied by agar gel electrophoresis in a variety of buffers. The highest maximal binding capacities of TBG and TBPA were found in Hank's balanced salt solution at pH 7.4. Phosphate and tris maleate ions at this pH resulted in the reduction of the thyroxine maximal binding capacity of TBG and TBPA. It was also shown that pH, ionic constitution and molarity of the system affect the distribution of tracer T4125I among the specific protein carriers. Thyroxine binding inhibitors were shown to affect TBPA, TBA and TBG at a physiological pH like the effect described at other pH levels. The relative binding potency of TBPA for thyroxine:DNP:salicylate:penicillin was in a ratio of 670:250:10:1, respectively. An attempt to deal with the binding capacity data by the Scatchard technique is described.