The binding of thyroxine to proteins in the blood plasma of the turtle, Trachemys scripta, was analyzed by incubation with radioactive thyroxine, electrophoresis and autoradiography. Albumin and an alpha-globulin were found to bind thyroxine; no thyroxine-binding transthyretin was detected in the prealbumin region. In contrast to blood plasma, a thyroxine-binding prealbumin was observed in medium from T. scripta choroid plexus incubated in vitro. RNA was extracted from brain tissue containing choroid plexus and from liver of T. scripta and Chelydra serpentina and analyzed by hybridization with transthyretin cDNA from the lizard Tiliqua rugosa. The brain RNAs contained substantial amounts of transthyretin mRNA, whereas only trace amounts of transthyretin mRNA were detected in RNA from liver. No transthyretin mRNA was observed in RNA from kidney. The results support the hypothesis that the expression of the transthyretin gene first evolved in the choroid plexus of the brain at the stage of the stem reptiles, whereas abundant transthyretin synthesis in liver evolved much later, and independently, in mammals and birds.