The patterns and the binding capacities of thyroxine-binding plasma proteins in various species of vertebrates were compared, and their evolutionary aspects were considered. Protein association of radioactivity in plasma-radioactive thyroxine mixtures was characterized by use of a radioautographic technique after cellulose acetate electrophoresis. Thyroxine-binding capacity of alpha-globulin was determined after adding carrier l-thyroxine to the plasma of the test animals: In Primates, prealbumin, albumin, and alpha-globulin-bound thyroxine; in cattle, albumin, post-albumin, and alpha-globulin-bound thyroxine; in other Artiodactyla, i.e., goats, sheep, muntjak deer, and swine, albumin- and alpha-globulin-bound thyroxine. In the Artiodactylas, the thyroxine-binding capacity by alpha-globulin was high compared with other orders of Mammalia and the lower classes of vertebrates. In horses and dogs, most radiothyroxine was bound to albumin and a relatively smaller amount of radioactivity was found in alpha-globulin. In cats and rabbits, the presence of thyroxine-binding alpha-globulin was questionable, and relatively strong binding of radiothyroxine was found in post-albumin and albumin. No apparent thyroxine-binding alpha-globulin was detectable in Rodentia. Bound radioactivity was found only in plasma albumin in guinea pigs, but in mice both in albumin and post-albumin. In rats, most radiothyroxine was bound to albumin and a very little amount of radioactivity was found in postalbumin. In lower vertebrates such as Aves, Reptilia, Amphibia, and Pisces, no thyroxine-binding alpha-globulin was found. Radiothyroxine concentrated in plasma albumin in some species (chicken, duck, caiman, fish), but in other species (pigeon, snake, lizard, tortoise, frog, newt), the radiothyroxine concentrated both in albumin and pre-albumin.